Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer

ABSTRACT

A novel gene 0193P1E1B (also designated 193P1E1B) and its encoded protein, and variants thereof, are described wherein 193P1E1B exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 193P1E1B provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 193P1E1B gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 193P1E1B can be used in active or passive immunization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from pending U.S. patent applicationSer. No. 10/013,312, filed 7 Dec. 2001. The contents of the applicationlisted in this paragraph is fully incorporated by reference herein.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention described herein relates to a gene and its encodedprotein, termed 193P1E1B, expressed in certain cancers, and todiagnostic and therapeutic methods and compositions useful in themanagement of cancers that express 193P1E1B.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, as reported by the American Cancer Society,cancer causes the death of well over a half-million people annually,with over 1.2 million new cases diagnosed per year. While deaths fromheart disease have been declining significantly, those resulting fromcancer generally are on the rise. In the early part of the next century,cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment.Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most commoncancer in males and is the second leading cause of cancer death in men.In the United States alone, well over 30,000 men die annually of thisdisease—second only to lung cancer. Despite the magnitude of thesefigures, there is still no effective treatment for metastatic prostatecancer. Surgical prostatectomy, radiation therapy, hormone ablationtherapy, surgical castration and chemotherapy continue to be the maintreatment modalities. Unfortunately, these treatments are ineffectivefor many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that canaccurately detect early-stage, localized tumors remains a significantlimitation in the diagnosis and management of this disease. Although theserum prostate specific antigen (PSA) assay has been a very useful tool,however its specificity and general utility is widely regarded aslacking in several important respects.

Progress in identifying additional specific markers for prostate cancerhas been improved by the generation of prostate cancer xenografts thatcan recapitulate different stages of the disease in mice. The LAPC (LosAngeles Prostate Cancer) xenografts are prostate cancer xenografts thathave survived passage in severe combined immune deficient (SCID) miceand have exhibited the capacity to mimic the transition from androgendependence to androgen independence (Klein et al., 1997, Nat. Med.3:402). More recently identified prostate cancer markers include PCTA-1(Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252),prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer ResSep. 2, 1996 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad SciUSA. 1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA)(Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA havefacilitated efforts to diagnose and treat prostate cancer, there is needfor the identification of additional markers and therapeutic targets forprostate and related cancers in order to further improve diagnosis andtherapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adultmalignancies. Once adenomas reach a diameter of 2 to 3 cm, malignantpotential exists. In the adult, the two principal malignant renal tumorsare renal cell adenocarcinoma and transitional cell carcinoma of therenal pelvis or ureter. The incidence of renal cell adenocarcinoma isestimated at more than 29,000 cases in the United States, and more than11,600 patients died of this disease in 1998. Transitional cellcarcinoma is less frequent, with an incidence of approximately 500 casesper year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma formany decades. Until recently, metastatic disease has been refractory toany systemic therapy. With recent developments in systemic therapies,particularly immunotherapies, metastatic renal cell carcinoma may beapproached aggressively in appropriate patients with a possibility ofdurable responses. Nevertheless, there is a remaining need for effectivetherapies for these patients.

Of all new cases of cancer in the United States, bladder cancerrepresents approximately 5 percent in men (fifth most common neoplasm)and 3 percent in women (eighth most common neoplasm). The incidence isincreasing slowly, concurrent with an increasing older population. In1998, there was an estimated 54,500 cases, including 39,500 in men and15,000 in women. The age-adjusted incidence in the United States is 32per 100,000 for men and eight per 100,000 in women. The historicmale/female ratio of 3:1 may be decreasing related to smoking patternsin women. There were an estimated 11,000 deaths from bladder cancer in1998 (7,800 in men and 3,900 in women). Bladder cancer incidence andmortality strongly increase with age and will be an increasing problemas the population becomes more elderly.

Most bladder cancers recur in the bladder. Bladder cancer is managedwith a combination of transurethral resection of the bladder (TUR) andintravesical chemotherapy or immunotherapy. The multifocal and recurrentnature of bladder cancer points out the limitations of TUR. Mostmuscle-invasive cancers are not cured by TUR alone. Radical cystectomyand urinary diversion is the most effective means to eliminate thecancer but carry an undeniable impact on urinary and sexual function.There continues to be a significant need for treatment modalities thatare beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in theUnited States, including 93,800 cases of colon cancer and 36,400 ofrectal cancer. Colorectal cancers are the third most common cancers inmen and women. Incidence rates declined significantly during 1992-1996(−2.1% per year). Research suggests that these declines have been due toincreased screening and polyp removal, preventing progression of polypsto invasive cancers. There were an estimated 56,300 deaths (47,700 fromcolon cancer, 8,600 from rectal cancer) in 2000, accounting for about11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectalcancer, and for cancers that have not spread, it is frequently curative.Chemotherapy, or chemotherapy plus radiation, is given before or aftersurgery to most patients whose cancer has deeply perforated the bowelwall or has spread to the lymph nodes. A permanent colostomy (creationof an abdominal opening for elimination of body wastes) is occasionallyneeded for colon cancer and is infrequently required for rectal cancer.There continues to be a need for effective diagnostic and treatmentmodalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancerin 2000, accounting for 14% of all U.S. cancer diagnoses. The incidencerate of lung and bronchial cancer is declining significantly in men,from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s,the rate of increase among women began to slow. In 1996, the incidencerate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000,accounting for 28% of all cancer deaths. During 1992-1996, mortalityfrom lung cancer declined significantly among men (−1.7% per year) whilerates for women were still significantly increasing (0.9% per year).Since 1987, more women have died each year of lung cancer than breastcancer, which, for over 40 years, was the major cause of cancer death inwomen. Decreasing lung cancer incidence and mortality rates most likelyresulted from decreased smoking rates over the previous 30 years;however, decreasing smoking patterns among women lag behind those ofmen. Of concern, although the declines in adult tobacco use have slowed,tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by thetype and stage of the cancer and include surgery, radiation therapy, andchemotherapy. For many localized cancers, surgery is usually thetreatment of choice. Because the disease has usually spread by the timeit is discovered, radiation therapy and chemotherapy are often needed incombination with surgery. Chemotherapy alone or combined with radiationis the treatment of choice for small cell lung cancer; on this regimen,a large percentage of patients experience remission, which in some casesis long lasting. There is however, an ongoing need for effectivetreatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were expectedto occur among women in the United States during 2000. Additionally,about 1,400 new cases of breast cancer were expected to be diagnosed inmen in 2000. After increasing about 4% per year in the 1980s, breastcancer incidence rates in women have leveled off in the 1990s to about110.6 cases per 100,000.

In the U.S. alone, there were an estimated 41,200 deaths (40,800 women,400 men) in 2000 due to breast cancer. Breast cancer ranks second amongcancer deaths in women. According to the most recent data, mortalityrates declined significantly during 1992-1996 with the largest decreasesin younger women, both white and black. These decreases were probablythe result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient'spreferences, treatment of breast cancer may involve lumpectomy (localremoval of the tumor) and removal of the lymph nodes under the arm;mastectomy (surgical removal of the breast) and removal of the lymphnodes under the arm; radiation therapy; chemotherapy; or hormonetherapy. Often, two or more methods are used in combination. Numerousstudies have shown that, for early stage disease, long-term survivalrates after lumpectomy plus radiotherapy are similar to survival ratesafter modified radical mastectomy. Significant advances inreconstruction techniques provide several options for breastreconstruction after mastectomy. Recently, such reconstruction has beendone at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amountsof surrounding normal breast tissue may prevent the local recurrence ofthe DCIS. Radiation to the breast and/or tamoxifen may reduce the chanceof DCIS occurring in the remaining breast tissue. This is importantbecause DCIS, if left untreated, may develop into invasive breastcancer. Nevertheless, there are serious side effects or sequelae tothese treatments. There is, therefore, a need for efficacious breastcancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the UnitedStates in 2000. It accounts for 4% of all cancers among women and rankssecond among gynecologic cancers. During 1992-1996, ovarian cancerincidence rates were significantly declining. Consequent to ovariancancer, there were an estimated 14,000 deaths in 2000. Ovarian cancercauses more deaths than any other cancer of the female reproductivesystem.

Surgery, radiation therapy, and chemotherapy are treatment options forovarian cancer. Surgery usually includes the removal of one or bothovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus(hysterectomy). In some very early tumors, only the involved ovary willbe removed, especially in young women who wish to have children. Inadvanced disease, an attempt is made to remove all intra-abdominaldisease to enhance the effect of chemotherapy. There continues to be animportant need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in theUnited States in 2000. Over the past 20 years, rates of pancreaticcancer have declined in men. Rates among women have remainedapproximately constant but may be beginning to decline. Pancreaticcancer caused an estimated 28,200 deaths in 2000 in the United States.Over the past 20 years, there has been a slight but significant decreasein mortality rates among men (about −0.9% per year) while rates haveincreased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options forpancreatic cancer. These treatment options can extend survival and/orrelieve symptoms in many patients but are not likely to produce a curefor most. There is a significant need for additional therapeutic anddiagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION

The present invention relates to a gene, designated 193P1 E1 B, that hasnow been found to be over-expressed in the cancer(s) listed in Table I.Northern blot expression analysis of 193P1E1B gene expression in normaltissues shows a restricted expression pattern in adult tissues. Thenucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of193P1E1B are provided. The tissue-related profile of 193P1 E1B in normaladult tissues, combined with the over-expression observed in the tissueslisted in Table I, shows that 193P1 E1B is aberrantly over-expressed inat least some cancers, and thus serves as a useful diagnostic,prophylactic, prognostic, and/or therapeutic target for cancers of thetissue(s) such as those listed in Table I.

The invention provides polynucleotides corresponding or complementary toall or part of the 193P1E1B genes, mRNAs, and/or coding sequences,preferably in isolated form, including polynucleotides encoding193P1E1B-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80,85, 90, 95, 100 or more than 100 contiguous amino acids of a193P1E1B-related protein, as well as the peptides/proteins themselves;DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides oroligonucleotides complementary or having at least a 90% homology to the193P1E1B genes or mRNA sequences or parts thereof, and polynucleotidesor oligonucleotides that hybridize to the 193P1E1B genes, mRNAs, or to193P1E1B-encoding polynucleotides. Also provided are means for isolatingcDNAs and the genes encoding 193P1E1B. Recombinant DNA moleculescontaining 193P1E1B polynucleotides, cells transformed or transducedwith such molecules, and host-vector systems for the expression of193P1E1B gene products are also provided. The invention further providesantibodies that bind to 193P1E1B proteins and polypeptide fragmentsthereof, including polyclonal and monoclonal antibodies, murine andother mammalian antibodies, chimeric antibodies, humanized and fullyhuman antibodies, and antibodies labeled with a detectable marker ortherapeutic agent. In certain embodiments, there is a proviso that theentire nucleic acid sequence of FIG. 2 is not encoded and/or the entireamino acid sequence of FIG. 2 is not prepared. In certain embodiments,the entire nucleic acid sequence of FIG. 2 is encoded and/or the entireamino acid sequence of FIG. 2 is prepared, either of which are inrespective human unit dose forms.

The invention further provides methods for detecting the presence andstatus of 193P1E1B polynucleotides and proteins in various biologicalsamples, as well as methods for identifying cells that express 193P1E1B.A typical embodiment of this invention provides methods for monitoring193P1E1B gene products in a issue or hematology sample having orsuspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeuticcompositions and strategies for treating cancers that express 193P1E1Bsuch as cancers of tissues listed in Table I, including therapies aimedat inhibiting the transcription, translation, processing or function of193P1E1B as well as cancer vaccines. In one aspect, the inventionprovides compositions, and methods comprising them, for treating acancer that expresses 193P1E1B in a human subject wherein thecomposition comprises a carrier suitable for human use and a human unitdose of one or more than one agent that inhibits the production orfunction of 193P1E1B. Preferably, the carrier is a uniquely humancarrier. In another aspect of the invention, the agent is a moiety thatis immunoreactive with 193P1E1B protein. Non-limiting examples of suchmoieties include, but are not limited to, antibodies (such as singlechain, monoclonal, polyclonal, humanized, chimeric, or humanantibodies), functional equivalents thereof (whether naturally occurringor synthetic), and combinations thereof. The antibodies can beconjugated to a diagnostic or therapeutic moiety. In another aspect, theagent is a small molecule as defined herein.

In another aspect, the agent comprises one or more than one peptidewhich comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLAclass I molecule in a human to elicit a CTL response to 193P1E1B and/orone or more than one peptide which comprises a helper T lymphocyte (HTL)epitope which binds an HLA class II molecule in a human to elicit an HTLresponse. The peptides of the invention may be on the same or on one ormore separate polypeptide molecules. In a further aspect of theinvention, the agent comprises one or more than one nucleic acidmolecule that expresses one or more than one of the CTL or HTL responsestimulating peptides as described above. In yet another aspect of theinvention, the one or more than one nucleic acid molecule may express amoiety that is immunologically reactive with 193P1E1B as describedabove. The one or more than one nucleic acid molecule may also be, orencodes, a molecule that inhibits production of 193P1E1B. Non-limitingexamples of such molecules include, but are not limited to, thosecomplementary to a nucleotide sequence essential for production of193P1E1B (e.g. antisense sequences or molecules that form a triple helixwith a nucleotide double helix essential for 193P1E1B production) or aribozyme effective to lyse 193P1E1B mRNA.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables)respective to its parental protein, e.g., variant 1, variant 2, etc.,reference is made to three factors: the particular variant, the lengthof the peptide in an HLA Peptide Table, and the Search Peptides in TableVII. Generally, a unique Search Peptide is used to obtain HLA peptidesof a particular for a particular variant. The position of each SearchPeptide relative to its respective parent molecule is listed in TableVII. Accordingly, if a Search Peptide begins at position “X”, one mustadd the value “X−1” to each position in Tables VIII-XXI and XXII to XLIXto obtain the actual position of the HLA peptides in their parentalmolecule. For example, if a particular Search Peptide begins at position150 of its parental molecule, one must add 150−1, i.e., 149 to each HLApeptide amino acid position to calculate the position of that amino acidin the parent molecule.

One embodiment of the invention comprises an HLA peptide, that occurs atleast twice in Tables VIII-XXI and XXII to XLIX collectively, or anoligonucleotide that encodes the HLA peptide. Another embodiment of theinvention comprises an HLA peptide that occurs at least once in TablesVIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotidethat encodes the HLA peptide.

Another embodiment of the invention is antibody epitopes, which comprisea peptide regions, or an oligonucleotide encoding the peptide region,that has one two, three, four, or five of the following characteristics:

i) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Hydrophilicity profile of FIG. 5;

ii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equalto 0.0, in the Hydropathicity profile of FIG. 6;

iii) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Percent Accessible Residues profile of FIG. 7;

iv) a peptide region of at least 5 amino acids of a particular peptideof FIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Average Flexibility profile of FIG. 8; or

v) a peptide region of at least 5 amino acids of a particular peptide ofFIG. 3, in any whole number increment up to the full length of thatprotein in FIG. 3, that includes an amino acid position having a valueequal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a valueequal to 1.0, in the Beta-turn profile of FIG. 9.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The 193P1E1B SSH sequence of 227 nucleotides.

FIG. 2. A) The cDNA and amino acid sequence of 193P1E1B variant 1 (alsocalled “193P1E1B v.1” or “193P1E1B variant 1”) is shown in FIG. 2A. Thestart methionine is underlined. The open reading frame extends fromnucleic acid 805-2043 including the stop codon.

B) The cDNA and amino acid sequence of 193P1E1B variant 2 (also called“193P1E1B v.2”) is shown in FIG. 2B. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

C) The cDNA and amino acid sequence of 193P1E1B variant 3 (also called“193P1E1B v.3”) is shown in FIG. 2C. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

D) The cDNA and amino acid sequence of 193P1E1B variant 4 (also called“193P1E1B v.4”) is shown in FIG. 2D. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

E) The cDNA and amino acid sequence of 193P1E1B variant 5 (also called“193P1E1B v.5”) is shown in FIG. 2E. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

F) The cDNA and amino acid sequence of 193P1E1B variant 6 (also called“193P1E1B v.6”) is shown in FIG. 2F. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

G) The cDNA and amino acid sequence of 193P1E1B variant 7 (also called“193P1E1B v.7”) is shown in FIG. 2G. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

H) The cDNA and amino acid sequence of 193P1E1B variant 8 (also called“193P1E1B v.8”) is shown in FIG. 2H. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-2043including the stop codon.

I) The cDNA and amino acid sequence of 193P1E1B variant 9 (also called“193P1E1B v.9”) is shown in FIG. 21. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 989-1981including the stop codon.

J) The cDNA and amino acid sequence of 193P1E1B variant 10 (also called“193P1E1B v.10”) is shown in FIG. 2J. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-1971including the stop codon.

K) The cDNA and amino acid sequence of 193P1E1B variant 11 (also called“193P1E1B v.11”) is shown in FIG. 2K. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 989-1909including the stop codon.

L) The cDNA and amino acid sequence of 193P1E1B variant 12 (also called“193P1E1B v.12”) is shown in FIG. 2L. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 805-1026including the stop codon.

M) The cDNA and amino acid sequence of 193P1E1B variant 13 (also called“193P1E1B v.13”) is shown in FIG. 2M. The codon for the start methionineis underlined. The open reading frame extends from nucleic acid 952-2070including the stop codon.

FIG. 3.

A) Amino acid sequence of 193P1E1B v.1 is shown in FIG. 3A; it has 412amino acids.

B) The amino acid sequence of 193P1E1B v.5 is shown in FIG. 3B; it has412 amino acids.

C) The amino acid sequence of 193P1E1B v.6 is shown in FIG. 3C; it has412 amino acids.

D) The amino acid sequence of 193P1E1B v.9 is shown in FIG. 3D; it has330 amino acids.

E) The amino acid sequence of 193P1E1B v.10 is shown in FIG. 3E; it has388 amino acids.

F) The amino acid sequence of 193P1E1B v.11 is shown in FIG. 3F; it has308 amino acids.

G) The amino acid sequence of 193P1E1B v.12 is shown in FIG. 3G; it has73 amino acids.

H) The amino acid sequence of 193P1E1B v.13 is shown in FIG. 3H; it has372 amino acids. As used herein, a reference to 193P1E1B includes allvariants thereof, including those shown in FIGS. 2, 3, 10, and 11,unless the context clearly indicates otherwise.

FIG. 4. FIG. 4A shows the alignment of 193P1E1B v.1 with gi 2178775.FIG. 4B shows the alignment of 193P1E1B v.5 with gi 2178775. FIG. 4Cshows the alignment of 193P1E1B v.11 with gi 2178775. FIG. 4D shows thealignment of 193P1E1B v.12 with gi 2178775. FIG. 4E shows the alignmentof 193P1E1B v.1 with E. coli arginine repressor. FIG. 4F shows theAlignment of 193P1E1B v.1 with human adenosine deaminase. FIG. 4G showsthe Clustal alignment of 193P1E1B protein variants.

FIG. 5. Hydrophilicity amino acid profile of 193P1E1B determined bycomputer algorithm sequence analysis using the method of Hopp and Woods(Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828) accessed on the Protscale website located on the WorldWide Web through the ExPasy molecular biology server.

FIG. 6. Hydropathicity amino acid profile of 193P1E1B determined bycomputer algorithm sequence analysis using the method of Kyte andDoolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)accessed on the ProtScale website located on the World Wide Web throughthe ExPasy molecular biology server.

FIG. 7. Percent accessible residues amino acid profile of 193P1E1Bdetermined by computer algorithm sequence analysis using the method ofJanin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScalewebsite located on the World Wide Web through the ExPasy molecularbiology server.

FIG. 8. Average flexibility amino acid profile of 193P1E1B determined bycomputer algorithm sequence analysis using the method of Bhaskaran andPonnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept.Protein Res. 32:242-255) accessed on the ProtScale website located onthe World Wide Web through the ExPasy molecular biology server.

FIG. 9. Beta-turn amino acid profile of 193P1E1B determined by computeralgorithm sequence analysis using the method of Deleage and Roux(Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed onthe ProtScale website located on the World Wide Web through the ExPasymolecular biology server.

FIG. 10. Schematic alignment of SNP variants of 193P1E1B. Variants193P1E1B v.2 through v.8 are variants with single nucleotidedifferences. Though these SNP variants are shown separately, they couldalso occur in any combinations and in any transcript variants thatcontains the base pairs. Numbers correspond to those of 193P1E1B v.1.Black box shows the same sequence as 193P1E1B v. 1. SNPs are indicatedabove the box.

FIG. 11. Schematic alignment of protein variants of 193P1E1B. Proteinvariants correspond to nucleotide variants. Nucleotide variants 193P1E1Bv.2, v.3, v.4, v.7, and v.8 in FIG. 10 code for the same protein as193P1E1B v.1. Nucleotide variants 193P1E1B v.9 through v.13 are splicevariants of v.1. Single amino acid differences were indicated above theboxes. Black boxes represent the same sequence as 193P1E1B v.1. Numbersunderneath the box correspond to amino acid positions in 193P1E1B v.1.

FIG. 12. Intentionally Omitted.

FIG. 13. Secondary structure prediction for 193P1E1B (SEQ ID NO: 123).The secondary structure of 193P1E1B protein was predicted using theHNN-Hierarchical Neural Network method , accessed from the ExPasymolecular biology server. This method predicts the presence and locationof alpha helices, extended strands, and random coils from the primaryprotein sequence. The percent of the protein in a given secondarystructure is as follows: h: Alpha helix 29.13%; c: Random coil 60.92%;e: Extended strand 9.95%.

FIG. 14. Expression of 193P1E1B by RT-PCR. (A) The schematic diagramdepicts the location of PCR primers Set A and set B on the sequences ofthe 3 variants of 193P1E1B. (B and C) First strand cDNA was preparedfrom vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2,pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool,bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancerpool, ovary cancer pool, breast cancer pool, metastasis cancer pool,pancreas cancer pool, and from prostate cancer metastasis to lymph nodefrom 2 different patients. Normalization was performed by PCR usingprimers to actin and GAPDH. Semi-quantitative PCR, using primer Set A(B) or primer Set B (C) to 193P1E1B, was performed at 30 cycles ofamplification. Strong expression of 193P1E1B was observed in prostatecancer xenograft pool, bladder cancer pool, kidney cancer pool, coloncancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,metastasis pool, pancreas cancer pool, and in the 2 different prostatecancer metastasis to lymph node. Low expression was observed in prostatecancer pool, but no expression was detected in VP1 and VP2. FIG. 14Cshows that the transcipt encoding encoding 193P1E1B v. 1 through v.8, isexpressed ate higher levels that the transcript encoding 193P1E1B v.9.But both transcripts are expressed at similar proportion in all tissuestested.

FIG. 15. Expression of 193P1E1B in normal human tissues. Two multipletissue northern blots, with 2 μg of mRNA/lane were probed with 193P1E1Bsequence. Size standards in kilobases (kb) are indicated on the side.The results show expression of two 193P1E1B transcripts, approximately3.5 kb and 2 kb, in testis and thymus.

FIG. 16. Expression of 193P1E1B in prostate cancer xenografts. RNA wasextracted from normal prostate, and from prostate cancer xenografts,LAPC-4AD, LAPC4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 μg oftotal RNA/lane was probed with 193P1E1B sequence. Size standards inkilobases (kb) are indicated on the side. The results show expression of193P1E1B in all 4 xenografts but not in normal prostate.

FIG. 17. Expression of 193P1E1B in patient cancer specimens. RNA wasextracted from a pool of three patients for each of the following,bladder cancer, colon cancer, ovary cancer and metastasis cancer, aswell as from normal prostate (NP), normal bladder (NB), normal kidney(NK), normal colon (NC). Northern blots with 10 μg of total RNA/lanewere probed with 193P1E1B sequence. Size standards in kilobases (kb) areindicated on the side. The results show expression of 193P1E1B inbladder cancer pool, colon cancer pool, ovary cancer pool and metastasiscancer pool, but not in any of the normal tissues tested.

FIG. 18. Expression of 193P1E1B in bladder cancer patient specimens. RNAwas extracted from bladder cancer cell lines (CL), normal bladder (N),bladder tumors (T) and matched normal adjacent tissue (NAT) isolatedfrom bladder cancer patients. Northern blots with 10 μg of totalRNA/lane were probed with 193P1E1B sequence. Size standards in kilobases(kb) are indicated on the side. The results show expression of 193P1E1Bin the two bladder cancer cell lines, and in 3 patient bladder tumorstested but not in normal bladder tissues.

FIG. 19. Expression of 193P1E1B in cancer metastasis patient specimens.RNA was extracted from the following cancer metastasis tissues, colonmetastasis to lung, lung metastasis to lymph node, lung metastasis toskin, and breast metastasis to lymph node, as well as from normalbladder (NB), normal lung (NL), normal breast (NBr), and normal ovary(NO). Northern blots with 10 μg of total RNA/lane were probed with193P1E1B sequence. Size standards in kilobases (kb) are indicated on theside. The results show expression of 193P1E1B in all four differentcancer metastasis samples but not in normal tissues.

FIG. 20. Expression of 193P1E1B in pancreas, ovary and testis cancerpatient specimens. RNA was extracted from pancreatic cancer (P1),ovarian cancer (P2, P3), and testicular cancer (P4, P5) isolated fromcancer patients, as well as from normal pancreas (NPa). Northern blotswith 10 μg of total RNA/lane were probed with 193P1E1B sequence. Sizestandards in kilobases (kb) are indicated on the side. The results showexpression of 193P1E1B in pancreatic, ovarian and testicular cancerspecimens but not in normal pancreas.

FIG. 21. Expression of 193P1E1B in Normal versus Patient CancerSpecimens. First strand cDNA was prepared from a panel of normal tissues(stomach, brain, heart, liver, spleen, skeletal muscle, tests prostate,bladder, kidney, colon, lung and pancreas) and from a panel of patientcancer pools (prostate cancer pool, bladder cancer pool, kidney cancerpool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovarycancer pool, breast cancer pool, metastasis cancer pool, LAPC prostatexenograft pool (XP), and from prostate cancer metastasis to lymph nodefrom 2 different patients (PMLN2). Normalization was performed by PCRusing primers to actin. Semi-quantitative PCR, using primer Set A asdescribed in FIG. 14, was performed was performed at 26 and 30 cycles ofamplification. Samples were run on an agarose gel, and PCR products werequantitated using the Alphalmager software. Relative expression wascalculated by normalizing to signal obtained using actin primers.Results show restricted 193P1E1B expression in normal testis amongst allnormal tissues tested. 193P1E1B expression was strongly upregulated incancers of the bladder, colon, lung, pancreas, ovary, breast, and to alesser extent in prostate and kidney cancers.

FIG. 22. Expression of 193P1E1B in Normal versus Patient CancerSpecimens. First strand cDNA was prepared from a panel of normal tissues(stomach, brain, heart, liver, spleen, skeletal muscle, testis prostate,bladder, kidney, colon, lung and pancreas) and from a panel of patientcancer pools (prostate cancer pool, bladder cancer pool, kidney cancerpool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovarycancer pool, breast cancer pool, metastasis cancer pool, LAPC prostatexenograft pool (XP), and from prostate cancer metastasis to lymph nodefrom 2 different patients (PMLN2). Normalization was performed by PCRusing primers to actin. Semi-quantitative PCR, using primer Set A asdescribed in FIG. 14, was performed was performed at 26 and 30 cycles ofamplification. Samples were run on an agarose gel, and PCR products werequantitated using the Alphalmager software. Relative expression wascalculated by normalizing to signal obtained using actin primers.Results show restricted 193P1E1B expression in normal testis amongst allnormal tissues tested. 193P1E1B expression was strongly upregulated incancers of the bladder, colon, lung, pancreas, ovary, breast, and to alesser extent in prostate and kidney cancers.

DETAILED DESCRIPTION OF THE INVENTION

Outline of Sections

I.) Definitions

II.) 193P1E1B Polynucleotides

II.A.) Uses of 193P1E1B Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

II.A.2.) Antisense Embodiments

II.A.3.) Primers and Primer Pairs

II.A.4.) Isolation of 193P1E1B-Encoding Nucleic Acid Molecules

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

III.) 193P1E1B-related Proteins

-   -   III.A.) Motif-bearing Protein Embodiments    -   III.B.) Expression of 193P1E1B-related Proteins    -   III.C.) Modifications of 193P1E1B-related Proteins    -   III.D.) Uses of 193P1E1B-related Proteins

IV.) 193P1E1B Antibodies

V.) 193P1E1B Cellular Immune Responses

VI.) 193P1E1B Transgenic Animals

VII.) Methods for the Detection of 193P1E1B

VIII.) Methods for Monitoring the Status of 193P1E1B-related Genes andTheir Products

IX.) Identification of Molecules That Interact With 193P1E1B

X.) Therapeutic Methods and Compositions

X.A.) Anti-Cancer Vaccines

X.B.) 193P1E1B as a Target for Antibody-Based Therapy

X.C.) 193P1E1B as a Target for Cellular Immune Responses

-   -   X.C.1. Minigene Vaccines    -   X.C.2. Combinations of CTL Peptides with Helper Peptides    -   X.C.3. Combinations of CTL Peptides with T Cell Priming Agents    -   X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or        HTL Peptides    -   X.D.) Adoptive Immunotherapy

X.E.) Administration of Vaccines for Therapeutic or ProphylacticPurposes

XI.) Diagnostic and Prognostic Embodiments of 193P1E1B.

XII.) Inhibition of 193P1E1B Protein Function

-   -   XII.A.) Inhibition of 193P1E1B With Intracellular Antibodies    -   XII.B.) Inhibition of 193P1E1B with Recombinant Proteins    -   XII.C.) Inhibition of 193P1E1B Transcription or Translation    -   XII.D.) General Considerations for Therapeutic Strategies

XIII.) Identification, Characterization and Use of Modulators of193P1E1B

XIV.) KITS/Articles of Manufacture

I.) Definitions:

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art, such as, forexample, the widely utilized molecular cloning methodologies describedin Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Asappropriate, procedures involving the use of commercially available kitsand reagents are generally carried out in accordance with manufacturerdefined protocols and/or parameters unless otherwise noted.

The terms “advanced prostate cancer”, “locally advanced prostatecancer”, “advanced disease” and “locally advanced disease” mean prostatecancers that have extended through the prostate capsule, and are meantto include stage C disease under the American Urological Association(AUA) system, stage C1-C2 disease under the Whitmore-Jewell system, andstage T3-T4 and N+ disease under the TNM (tumor, node, metastasis)system. In general, surgery is not recommended for patients with locallyadvanced disease, and these patients have substantially less favorableoutcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

“Altering the native glycosylation pattern” is intended for purposesherein to mean deleting one or more carbohydrate moieties found innative sequence 193P1E1B (either by removing the underlyingglycosylation site or by deleting the glycosylation by chemical and/orenzymatic means), and/or adding one or more glycosylation sites that arenot present in the native sequence 193P1E1B. In addition, the phraseincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present.

The term “analog” refers to a molecule which is structurally similar orshares similar or corresponding attributes with another molecule (e.g. a193P1E1B-related protein). For example, an analog of a 193P1E1B proteincan be specifically bound by an antibody or T cell that specificallybinds to 193P1E1B.

The term “antibody” is used in the broadest sense. Therefore, an“antibody” can be naturally occurring or man-made such as monoclonalantibodies produced by conventional hybridoma technology. Anti-193P1E1Bantibodies comprise monoclonal and polyclonal antibodies as well asfragments containing the antigen-binding domain and/or one or morecomplementarity determining regions of these antibodies.

An “antibody fragment” is defined as at least a portion of the variableregion of the immunoglobulin molecule that binds to its target, i.e.,the antigen-binding region. In one embodiment it specifically coverssingle anti-193P1E1B antibodies and clones thereof (including agonist,antagonist and neutralizing antibodies) and anti-193P1E1B antibodycompositions with polyepitopic specificity.

The term “codon optimized sequences” refers to nucleotide sequences thathave been optimized for a particular host species by replacing anycodons having a usage frequency of less than about 20%. Nucleotidesequences that have been optimized for expression in a given hostspecies by elimination of spurious polyadenylation sequences,elimination of exon/intron splicing signals, elimination oftransposon-like repeats and/or optimization of GC content in addition tocodon optimization are referred to herein as an “expression enhancedsequences.”

A “combinatorial library” is a collection of diverse chemical compoundsgenerated by either chemical synthesis or biological synthesis bycombining a number of chemical “building blocks” such as reagents. Forexample, a linear combinatorial chemical library, such as a polypeptide(e.g., mutein) library, is formed by combining a set of chemicalbuilding blocks called amino acids in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Numerous chemical compounds are synthesized through suchcombinatorial mixing of chemical building blocks (Gallop et al., J. Med.Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known tothose of skill in the art. Such combinatorial chemical librariesinclude, but are not limited to, peptide libraries (see, e.g., U.S. Pat.No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton etal., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat.No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with a Beta-D-Glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbarnates (Cho, etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J.Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g.,Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat.No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., NatureBiotechnology 14(3): 309-314 (1996), and PCT/US96110287), carbohydratelibraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), andU.S. Pat. No. 5,593,853), and small organic molecule libraries (see,e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids,U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat.No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S.Pat. No. 5,288,514; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, FosterCity, Calif.; 9050, Plus, Millipore, Bedford, Mass.). A number ofwell-known robotic systems have also been developed for solution phasechemistries. These systems include automated workstations such as theautomated synthesis apparatus developed by Takeda Chemical Industries,LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms(Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard,Palo Alto, Calif.), which mimic the manual synthetic operationsperformed by a chemist. Any of the above devices are suitable for usewith the present invention. The nature and implementation ofmodifications to these devices (if any) so that they can operate asdiscussed herein will be apparent to persons skilled in the relevantart. In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex,Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

The term “cytotoxic agent” refers to a substance that inhibits orprevents the expression activity of cells, function of cells and/orcauses destruction of cells. The term is intended to include radioactiveisotopes chemotherapeutic agents, and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Examples ofcytotoxic agents include, but are not limited to auristatins,auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain,combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin,taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40,abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin,mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin,calicheamicin, Sapaonana officinalis inhibitor, and glucocorticoid andother chemotherapeutic agents, as well as radioisotopes such as At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi^(212 or 213), P³² andradioactive isotopes of Lu including Lu¹⁷⁷. Antibodies may also beconjugated to an anti-cancer pro-drug activating enzyme capable ofconverting the pro-drug to its active form.

The “gene product” is sometimes referred to herein as a protein or mRNA.For example, a “gene product of the invention” is sometimes referred toherein as a “cancer amino acid sequence”, “cancer protein”, “protein ofa cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listedin Table I”, etc. In one embodiment, the cancer protein is encoded by anucleic acid of FIG. 2. The cancer protein can be a fragment, oralternatively, be the full-length protein to the fragment encoded by thenucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequenceis used to determine sequence identity or similarity. In anotherembodiment, the sequences are naturally occurring allelic variants of aprotein encoded by a nucleic acid of FIG. 2. In another embodiment, thesequences are sequence variants as further described herein.

“High throughput screening” assays for the presence, absence,quantification, or other properties of particular nucleic acids orprotein products are well known to those of skill in the art. Similarly,binding assays and reporter gene assays are similarly well known. Thus,e.g., U.S. Pat. No. 5,559,410 discloses high throughput screeningmethods for proteins; U.S. Pat. No. 5,585,639 discloses high throughputscreening methods for nucleic acid binding (i.e., in arrays); while U.S.Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods ofscreening for ligand/antibody binding.

In addition, high throughput screening systems are commerciallyavailable (see, e.g., Amersham Biosciences, Piscataway, N.J.; ZymarkCorp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; BeckmanInstruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick,Mass.; etc.). These systems typically automate entire procedures,including all sample and reagent pipetting, liquid dispensing, timedincubations, and final readings of the microplate in detector(s)appropriate for the assay. These configurable systems provide highthroughput and rapid start up as well as a high degree of flexibilityand customization. The manufacturers of such systems provide detailedprotocols for various high throughput systems. Thus, e.g., Zymark Corp.provides technical bulletins describing screening systems for detectingthe modulation of gene transcription, ligand binding, and the like.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

“Human Leukocyte Antigen” or “HLA” is a human class I or class II MajorHistocompatibility Complex (MHC) protein (see, e.g., Stites, et al.,IMMUNOLOGY, 8^(TH) ED., Lange Publishing, Los Altos, Calif. (1994).

The terms “hybridize”, “hybridizing”, “hybridizes” and the like, used inthe context of polynucleotides, are meant to refer to conventionalhybridization conditions, preferably such as hybridization in 50%formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures forhybridization are above 37 degrees C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55 degrees C.

The phrases “isolated” or “biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state. Thus,isolated peptides in accordance with the invention preferably do notcontain materials normally associated with the peptides in their in situenvironment. For example, a polynucleotide is said to be “isolated” whenit is substantially separated from contaminant polynucleotides thatcorrespond or are complementary to genes other than the 193P1E1B genesor that encode polypeptides other than 193P1E1B gene product orfragments thereof. A skilled artisan can readily employ nucleic acidisolation procedures to obtain an isolated 193P1E1B polynucleotide. Aprotein is said to be “isolated,” for example, when physical, mechanicalor chemical methods are employed to remove the 193P1E1B proteins fromcellular constituents that are normally associated with the protein. Askilled artisan can readily employ standard purification methods toobtain an isolated 193P1E1B protein. Alternatively, an isolated proteincan be prepared by chemical means.

The term “mammal” refers to any organism classified as a mammal,including mice, rats, rabbits, dogs, cats, cows, horses and humans. Inone embodiment of the invention, the mammal is a mouse. In anotherembodiment of the invention, the mammal is a human.

The terms “metastatic prostate cancer” and “metastatic disease” meanprostate cancers that have spread to regional lymph nodes or to distantsites, and are meant to include stage D disease under the AUA system andstage TxNxM+ under the TNM system. As is the case with locally advancedprostate cancer, surgery is generally not indicated for patients withmetastatic disease, and hormonal (androgen ablation) therapy is apreferred treatment modality. Patients with metastatic prostate cancereventually develop an androgen-refractory state within 12 to 18 monthsof treatment initiation. Approximately half of these androgen-refractorypatients die within 6 months after developing that status. The mostcommon site for prostate cancer metastasis is bone. Prostate cancer bonemetastases are often osteoblastic rather than osteolytic (i.e.,resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

The term “modulator” or “test compound” or “drug candidate” orgrammatical equivalents as used herein describe any molecule, e.g.,protein, oligopeptide, small organic molecule, polysaccharide,polynucleotide, etc., to be tested for the capacity to directly orindirectly alter the cancer phenotype or the expression of a cancersequence, e.g., a nucleic acid or protein sequences, or effects ofcancer sequences (e.g., signaling, gene expression, protein interaction,etc.) In one aspect, a modulator will neutralize the effect of a cancerprotein of the invention. By “neutralize” is meant that an activity of aprotein is inhibited or blocked, along with the consequent effect on thecell. In another aspect, a modulator will neutralize the effect of agene, and its corresponding protein, of the invention by normalizinglevels of said protein. In preferred embodiments, modulators alterexpression profiles, or expression profile nucleic acids or proteinsprovided herein, or downstream effector pathways. In one embodiment, themodulator suppresses a cancer phenotype, e.g. to a normal tissuefingerprint. In another embodiment, a modulator induced a cancerphenotype. Generally, a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e., at zero concentration or below thelevel of detection.

Modulators, drug candidates or test compounds encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 100 and less than about 2,500 Daltons. Preferred small moleculesare less than 2000, or less than 1500 or less than 1000 or less than 500D. Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Modulators also comprise biomolecules such aspeptides, saccharides, fatty acids, steroids, purines, pyrimidines,derivatives, structural analogs or combinations thereof. Particularlypreferred are peptides. One class of modulators are peptides, forexample of from about five to about 35 amino acids, with from about fiveto about 20 amino acids being preferred, and from about 7 to about 15being particularly preferred. Preferably, the cancer modulatory proteinis soluble, includes a non-transmembrane region, and/or, has anN-terminal Cys to aid in solubility. In one embodiment, the C-terminusof the fragment is kept as a free acid and the N-terminus is a freeamine to aid in coupling, i.e., to cysteine. In one embodiment, a cancerprotein of the invention is conjugated to an immunogenic agent asdiscussed herein. In one embodiment, the cancer protein is conjugated toBSA. The peptides of the invention, e.g., of preferred lengths, can belinked to each other or to other amino acids to create a longerpeptide/protein. The modulatory peptides can be digests of naturallyoccurring proteins as is outlined above, random peptides, or “biased”random peptides. In a preferred embodiment, peptide/protein-basedmodulators are antibodies, and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulatingagents can be naturally occurring nucleic acids, random nucleic acids,or “biased” random nucleic acids. For example, digests of prokaryotic oreukaryotic genomes can be used in an approach analogous to that outlinedabove for proteins.

The term “monoclonal antibody” refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the antibodiescomprising the population are identical except for possible naturallyoccurring mutations that are present in minor amounts.

A“motif”, as in biological motif of a 193P1E1B-related protein, refersto any pattern of amino acids forming part of the primary sequence of aprotein, that is associated with a particular function (e.g.protein-protein interaction, protein-DNA interaction, etc) ormodification (e.g. that is phosphorylated, glycosylated or amidated), orlocalization (e.g. secretory sequence, nuclear localization sequence,etc.) or a sequence that is correlated with being immunogenic, eitherhumorally or cellularly. A motif can be either contiguous or capable ofbeing aligned to certain positions that are generally correlated with acertain function or property. In the context of HLA motifs, “motif”refers to the pattern of residues in a peptide of defined length,usually a peptide of from about 8 to about 13 amino acids for a class IHLA motif and from about 6 to about 25 amino acids for a class II HLAmotif, which is recognized by a particular HLA molecule. Peptide motifsfor HLA binding are typically different for each protein encoded by eachhuman HLA allele and differ in the patter of the primary and secondaryanchor residues.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” refers to a non-toxic, inert, and/orcomposition that is physiologically compatible with humans or othermammals.

The term “polynucleotide” means a polymeric form of nucleotides of atleast 10 bases or base pairs in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, and ismeant to include single and double stranded forms of DNA and/or RNA. Inthe art, this term if often used interchangeably with “oligonucleotide”.A polynucleotide can comprise a nucleotide sequence disclosed hereinwherein thymidine (T), as shown for example in FIG. 2, can also beuracil (U); this definition pertains to the differences between thechemical structures of DNA and RNA, in particular the observation thatone of the four major bases in RNA is uracil (U) instead of thymidine(T).

The term “polypeptide” means a polymer of at least about 4, 5, 6, 7, or8 amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used. In the art, thisterm is often used interchangeably with “peptide” or “protein”.

An HLA “primary anchor residue” is an amino acid at a specific positionalong a peptide sequence which is understood to provide a contact pointbetween the immunogenic peptide and the HLA molecule. One to three,usually two, primary anchor residues within a peptide of defined lengthgenerally defines a “motif” for an immunogenic peptide. These residuesare understood to fit in close contact with peptide binding groove of anHLA molecule, with their side chains buried in specific pockets of thebinding groove. In one embodiment, for example, the primary anchorresidues for an HLA class I molecule are located at position 2 (from theamino terminal position) and at the carboxyl terminal position of a 8,9, 10, 11, or 12 residue peptide epitope in accordance with theinvention. Alternatively, in another embodiment, the primary anchorresidues of a peptide binds an HLA class II molecule are spaced relativeto each other, rather than to the termini of a peptide, where thepeptide is generally of at least 9 amino acids in length. The primaryanchor positions for each motif and supermotif are set forth in TableIV. For example, analog peptides can be created by altering the presenceor absence of particular residues in the primary and/or secondary anchorpositions shown in Table IV. Such analogs are used to modulate thebinding affinity and/or population coverage of a peptide comprising aparticular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following(non-limiting exemplary uses are also set forth):

Examples of Medical Isotopes:

-   Isotope-   Description of use    Actinium-225-   (AC-225)-   See Thorium-229 (Th-229)    Actinium-227-   (AC-227)-   Parent of Radium-223 (Ra-223) which is an alpha emitter used to    treat metastases in the skeleton resulting from cancer (i.e., breast    and prostate cancers), and cancer radioimmunotherapy    Bismuth-212-   (Bi-212)-   See Thorium-228 (Th-228)    Bismuth-213-   (Bi-213)-   See Thorium-229 (Th-229)    Cadmium-109-   (Cd-109)-   Cancer detection    Cobalt-60-   (Co-60)-   Radiation source for radiotherapy of cancer, for food irradiators,    and for sterilization of medical supplies    Copper-64-   (Cu-64)-   A positron emitter used for cancer therapy and SPECT imaging    Copper-67-   (Cu-67)-   Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic    studies (i.e., breast and colon cancers, and lymphoma)    Dysprosium-166-   (Dy-166)-   Cancer radioimmunotherapy    Erbium-169-   (Er-169)-   Rheumatoid arthritis treatment, particularly for the small joints    associated with fingers and toes    Europium-152-   (Eu-152)-   Radiation source for food irradiation and for sterilization of    medical supplies    Europium-154-   (Eu-154)-   Radiation source for food irradiation and for sterilization of    medical supplies    Gadolinium-153-   (Gd-153)-   Osteoporosis detection and nuclear medical quality assurance devices    Gold-198-   (Au-198)-   Implant and intracavity therapy of ovarian, prostate, and brain    cancers    Holmium-166-   (Ho-166)-   Multiple myeloma treatment in targeted skeletal therapy, cancer    radioimmunotherapy, bone marrow ablation, and rheumatoid arthritis    treatment    Iodine-125-   (I-125)-   Osteoporosis detection, diagnostic imaging, tracer drugs, brain    cancer treatment, radiolabeling, tumor imaging, mapping of receptors    in the brain, interstitial radiation therapy, brachytherapy for    treatment of prostate cancer, determination of glomerular filtration    rate (GFR), determination of plasma volume, detection of deep vein    thrombosis of the legs    Iodine-131-   (I-131)-   Thyroid function evaluation, thyroid disease detection, treatment of    thyroid cancer as well as other non-malignant thyroid diseases    (i.e., Graves disease, goiters, and hyperthyroidism), treatment of    leukemia, lymphoma, and other forms of cancer (e.g., breast cancer)    using radioimmunotherapy    Iridium-192-   (Ir-192)-   Brachytherapy, brain and spinal cord tumor treatment, treatment of    blocked arteries (i.e., arteriosclerosis and restenosis), and    implants for breast and prostate tumors    Lutetium-177-   (Lu-177)-   Cancer radioimmunotherapy and treatment of blocked arteries (i.e.,    arteriosclerosis and restenosis)    Molybdenum-99-   (Mo-99)-   Parent of Technetium-99m (Tc-99m) which is used for imaging the    brain, liver, lungs, heart, and other organs. Currently, Tc-99m is    the most widely used radioisotope used for diagnostic imaging of    various cancers and diseases involving the brain, heart, liver,    lungs; also used in detection of deep vein thrombosis of the legs    Osmium-194-   (Os-194)-   Cancer radioimmunotherapy    Palladium-103-   (Pd-103)-   Prostate cancer treatment    Platinum-195m-   (Pt-195m)-   Studies on biodistribution and metabolism of cisplatin, a    chemotherapeutic drug    Phosphorus-32-   (P-32)-   Polycythemia rubra vera (blood cell disease) and leukemia treatment,    bone cancer diagnosis/treatment; colon, pancreatic, and liver cancer    treatment; radiolabeling nucleic acids for in vitro research,    diagnosis of superficial tumors, treatment of blocked arteries    (i.e., arteriosclerosis and restenosis), and intracavity therapy    Phosphorus-33-   (P-33)-   Leukemia treatment, bone disease diagnosis/treatment, radiolabeling,    and treatment of blocked arteries (i.e., arteriosclerosis and    restenosis)    Radium-223-   (Ra-223)-   See Actinium-227 (Ac-227)    Rhenium-186-   (Re-186)-   Bone cancer pain relief, rheumatoid arthritis treatment, and    diagnosis and treatment of lymphoma and bone, breast, colon, and    liver cancers using radioimmunotherapy    Rhenium-188-   (Re-188)-   Cancer diagnosis and treatment using radioimmunotherapy, bone cancer    pain relief, treatment of rheumatoid arthritis, and treatment of    prostate cancer    Rhodium-105-   (Rh-105)-   Cancer radioimmunotherapy    Samarium-145-   (Sm-145)-   Ocular cancer treatment    Samarium-153-   (Sm-153)-   Cancer radioimmunotherapy and bone cancer pain relief    Scandium-47-   (Sc-47)-   Cancer radioimmunotherapy and bone cancer pain relief    Selenium-75-   (Se-75)-   Radiotracer used in brain studies, imaging of adrenal cortex by    gamma-scintigraphy, lateral locations of steroid secreting tumors,    pancreatic scanning, detection of hyperactive parathyroid glands,    measure rate of bile acid loss from the endogenous pool    Strontium-85-   (Sr-85)-   Bone cancer detection and brain scans    Strontium-89-   (Sr-89)-   Bone cancer pain relief, multiple myeloma treatment, and    osteoblastic therapy    Technetium-99m-   (Tc-99m)-   See Molybdenum-99 (Mo-99)    Thorium-228-   (Th-228)-   Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in    cancer radioimmunotherapy    Thorium-229-   (Th-229)-   Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213    (Bi-213) which are alpha emitters used in cancer radioimmunotherapy    Thulium-170-   (Tm-170)-   Gamma source for blood irradiators, energy source for implanted    medical devices    Tin-117m-   (Sn-117m)-   Cancer immunotherapy and bone cancer pain relief    Tungsten-188-   (W-188)-   Parent for Rhenium-188 (Re-188) which is used for cancer    diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis    treatment, and treatment of blocked arteries (i.e., arteriosclerosis    and restenosis)    Xenon-127-   (Xe-127)-   Neuroimaging of brain disorders, high resolution SPECT studies,    pulmonary function tests, and cerebral blood flow studies    Ytterbium-175-   (Yb-175)-   Cancer radioimmunotherapy    Yttrium-90-   (Y-90)-   Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver    cancer treatment    Yttrium-91-   (Y-91)-   A gamma-emitting label for Yttrium-90 (Y-90) which is used for    cancer radioimmunotherapy (i.e., lymphoma, breast, colon, kidney,    lung, ovarian, prostate, pancreatic, and inoperable liver cancers)

By “randomized” or grammatical equivalents as herein applied to nucleicacids and proteins is meant that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. Theserandom peptides (or nucleic acids, discussed herein) can incorporate anynucleotide or amino acid at any position. The synthetic process can bedesigned to generate randomized proteins or nucleic acids, to allow theformation of all or most of the possible combinations over the length ofthe sequence, thus forming a library of randomized candidate bioactiveproteinaceous agents.

In one embodiment, a library is “fully randomized,” with no sequencepreferences or constants at any position. In another embodiment, thelibrary is a “biased random” library. That is, some positions within thesequence either are held constant, or are selected from a limited numberof possibilities. For example, the nucleotides or amino acid residuesare randomized within a defined class, e.g., of hydrophobic amino acids,hydrophilic residues, sterically biased (either small or large)residues, towards the creation of nucleic acid binding domains, thecreation of cysteines, for cross-linking, prolines for SH-3 domains,serines, threonines, tyrosines or histidines for phosphorylation sites,etc., or to purines, etc.

A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that-hasbeen subjected to molecular manipulation in vitro.

Non-limiting examples of small molecules include compounds that bind orinteract with 193P1E1B, ligands including hormones, neuropeptides,chemokines, odorants, phospholipids, and functional equivalents thereofthat bind and preferably inhibit 193P1E1B protein function. Suchnon-limiting small molecules preferably have a molecular weight of lessthan about 10 kDa, more preferably below about 9, about 8, about 7,about 6, about 5 or about 4 kDa. In certain embodiments, small moleculesphysically associate with, or bind, 193P1E1B protein; are not found innaturally occurring metabolic pathways; and/or are more soluble inaqueous than non-aqueous solutions

“Stringency” of hybridization reactions is readily determinable by oneof ordinary skill in the art, and generally is an empirical calculationdependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured nucleic acidsequences to reanneal when complementary strands are present in anenvironment below their melting temperature. The higher the degree ofdesired homology between the probe and hybridizable sequence, the higherthe relative temperature that can be used. As a result, it follows thathigher relative temperatures would tend to make the reaction conditionsmore stringent, while lower temperatures less so. For additional detailsand explanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

“Stringent conditions” or “high stringency conditions”, as definedherein, are identified by, but not limited to, those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at5° C.; (2) employ during hybridization a denaturing agent, such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 Msodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodiumpyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42°C. in 0.2 x SSC (sodium chloride/sodium. citrate) and 50% formamide at55° C., followed by a high-stringency wash consisting of 0.1 x SSCcontaining EDTA at 55° C. “Moderately stringent conditions” aredescribed by, but not limited to, those in Sambrook et al., MolecularCloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989,and include the use of washing solution and hybridization conditions(e.g., temperature, ionic strength and % SDS) less stringent than thosedescribed above. An example of moderately stringent conditions isovernight incubation at 37° C. in a solution comprising: 20% formamide,5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate(pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mLdenatured sheared salmon sperm DNA, followed by washing the filters in 1x SSC at about 37-50° C. The skilled artisan will recognize how toadjust the temperature, ionic strength, etc. as necessary to accommodatefactors such as probe length and the like.

An HLA “supermotif” is a peptide binding specificity shared by HLAmolecules encoded by two or more HLA alleles. Overall phenotypicfrequencies of HLA-supertypes in different ethnic populations are setforth in Table IV (F). The non-limiting constituents of varioussupetypes are as follows:

A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901,A*0207

A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101

B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701,B*7801, B*0702, B*5101, B*5602

B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)

A1: A*0102, A*2604, A*3601, A*4301, A*8001

A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003

B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901,B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08

B58: B*1516, B*1517, B*5701, B*5702, B58

B62: B*4601, 652, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Calculated population coverage afforded by different HLA-supertypecombinations are set forth in Table IV (G).

As used herein “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich are the byproducts of an alternative therapeutic modality; fulleradication of disease is not required.

A “transgenic animal” (e.g., a mouse or rat) is an animal having cellsthat contain a transgene, which transgene was introduced into the animalor an ancestor of the animal at a prenatal, e.g., an embryonic stage. A“transgene” is a DNA that is integrated into the genome of a cell fromwhich a transgenic animal develops.

As used herein, an HLA or cellular immune response “vaccine” is acomposition that contains or encodes one or more peptides of theinvention. There are numerous embodiments of such vaccines, such as acocktail of one or more individual peptides; one or more peptides of theinvention comprised by a polyepitopic peptide; or nucleic acids thatencode such individual peptides or polypeptides, e.g., a minigene thatencodes a polyepitopic peptide. The “one or more peptides” can includeany whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides ofthe invention. The peptides or polypeptides can optionally be modified,such as by lipidation, addition of targeting or other sequences. HLAclass I peptides of the invention can be admixed with, or linked to, HLAclass II peptides, to facilitate activation of both cytotoxic Tlymphocytes and helper T lymphocytes. HLA vaccines can also comprisepeptide-pulsed antigen presenting cells, e.g., dendritic cells.

The term “variant” refers to a molecule that exhibits a variation from adescribed type or norm, such as a protein that has one or more differentamino acid residues in the corresponding position(s) of a specificallydescribed protein (e.g. the 193P1E1B protein shown in FIG. 2 or FIG. 3.An analog is an example of a variant protein. Splice isoforms and singlenucleotides polymorphisms (SNPs) are further examples of variants.

The “193P1E1B-related proteins” of the invention include thosespecifically identified herein, as well as allelic variants,conservative substitution variants, analogs and homologs that can beisolated/generated and characterized without undue experimentationfollowing the methods outlined herein or readily available in the art.Fusion proteins that combine parts of different 193P1E1B proteins orfragments thereof, as well as fusion proteins of a 193P1E1B protein anda heterologous polypeptide are also included. Such 193P1E1B proteins arecollectively referred to as the 193P1E1B-related proteins, the proteinsof the invention, or 193P1E1B. The term “193P1E1B-related protein”refers to a polypeptide fragment or a 193P1E1B protein sequence of 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, or more than 25amino acids; or, at least 30, 35, 40, 45, 50, 55, 60,65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140,145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250,275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600,625, 650, or 664 or more amino acids.

II.) 193P1E1B Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a 193P1E1B gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a 193P1E1B-related protein and fragments thereof, DNA, RNA,DNA/RNA hybrid, and related molecules, polynucleotides oroligonucleotides complementary to a 193P1E1B gene or mRNA sequence or apart thereof, and polynucleotides or oligonucleotides that hybridize toa 193P1E1B gene, mRNA, or to a 193P1E1B encoding polynucleotide(collectively, “193P1E1B polynucleotides”). In all instances whenreferred to in this section, T can also be U in FIG. 2.

Embodiments of a 193P1E1B polynucleotide include: a 193P1E1Bpolynucleotide having the sequence shown in FIG. 2, the nucleotidesequence of 193P1E1B as shown in FIG. 2 wherein T is U; at least 10contiguous nucleotides of a polynucleotide having the sequence as shownin FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotidehaving the sequence as shown in FIG. 2 where T is U. For example,embodiments of 193P1E1B nucleotides comprise, without limitation:

-   -   (I) a polynucleotide comprising, consisting essentially of, or        consisting of a sequence as shown in FIG. 2, wherein T can also        be U;    -   (II) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2A, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the stop codon, wherein T can also be U;    -   (III) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2B, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the stop codon, wherein T can also be U;    -   (IV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2C, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the a stop codon, wherein T can also be U;    -   (V) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2D, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the stop codon, wherein T can also be U;    -   (VI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2E, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the stop codon, wherein T can also be U;    -   (VII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2F, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the stop codon, wherein T can also be U;    -   (VIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2G, from        nucleotide residue number 805 through nucleotide residue number        2043, including the stop codon, wherein T can also be U;    -   (IX) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2H, from nucleotide        residue number 805 through nucleotide residue number 2043,        including the stop codon, wherein T can also be U;    -   (X) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 21, from nucleotide        residue number 989 through nucleotide residue number 1981,        including the stop codon, wherein T can also be U;    -   (XI) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2J, from nucleotide        residue number 805 through nucleotide residue number 1971,        including the stop codon, wherein T can also be U;    -   (XII) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2K, from nucleotide        residue number 989 through nucleotide residue number 1909,        including the stop codon, wherein T can also be U;    -   (XIII) a polynucleotide comprising, consisting essentially of,        or consisting of the sequence as shown in FIG. 2L, from        nucleotide residue number 805 through nucleotide residue number        1026, including the stop codon, wherein T can also be U;    -   (XIV) a polynucleotide comprising, consisting essentially of, or        consisting of the sequence as shown in FIG. 2M, from nucleotide        residue number 952 through nucleotide residue number 2070,        including the stop codon, wherein T can also be U;    -   (XV) a polynucleotide that encodes a 193P1E1B-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        homologous to an entire amino acid sequence shown in FIGS. 2A-M;    -   (XVI) a polynucleotide that encodes a 193P1E1B-related protein        that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%        identical to an entire amino acid sequence shown in FIGS. 2A-M;    -   (XVII) a polynucleotide that encodes at least one peptide set        forth in Tables VIII-XXI and XXII-XLIX;    -   (XVIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-C in any whole number increment        up to 412 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,        11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,        27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having        a value greater than 0.5 in the Hydrophilicity profile of FIG.        5;    -   (XIX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-C in any whole number increment        up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-C in any whole number increment        up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXI) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-C in any whole number increment        up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIGS. 3A-C in any whole number increment        up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3D in any whole number increment up        to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3D in any whole number increment up        to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3D in any whole number increment up        to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3D in any whole number increment up        to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXVII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3D in any whole number increment up        to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XXVIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXIX) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXX) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXXI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXXII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3E in any whole number increment up        to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XXXIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXXIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XXXV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XXXVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33,,34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XXXVII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3F in any whole number increment up        to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XXXVIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XXXIX) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XL) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XLI) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XLII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3G in any whole number increment up        to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XLIII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3H in any whole number increment up        to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (XLIV) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3H in any whole number increment up        to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        less than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XLV) a polynucleotide that encodes a peptide region of at least        5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,        22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3H in any whole number increment up        to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XLVI) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3H in any whole number increment up        to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XLVII) a polynucleotide that encodes a peptide region of at        least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,        21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino        acids of a peptide of FIG. 3H in any whole number increment up        to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9    -   (XLVIII) a polynucleotide that is fully complementary to a        polynucleotide of any one of (I)-(XLVII). (XLIX) a peptide that        is encoded by any of (I) to (XLVIII); and    -   (L) a composition comprising a polynucleotide of any of        (I)-(XLVIII) or peptide of (XLIX) together with a pharmaceutical        excipient and/or in a human unit dose form.    -   (LI) a method of using a polynucleotide of any (I)-(XLVIII) or        peptide of (XLIX) or a composition of (L) in a method to        modulate a cell expressing 193P1E1B,    -   (LII) a method of using a polynucleotide of any (I)-(XLVIII) or        peptide of (XLIX) or a composition of (L) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing 193P1E1B    -   (LIII) a method of using a polynucleotide of any (I)-(XLVIII) or        peptide of (XLIX) or a composition of (L) in a method to        diagnose, prophylax, prognose, or treat an individual who bears        a cell expressing 193P1E1B, said cell from a cancer of a tissue        listed in Table I;    -   (LIV) a method of using a polynucleotide of any (I)-(XLVIII) or        peptide of (XLIX) or a composition of (L) in a method to        diagnose, prophylax, prognose, or treat a a cancer;    -   (LV) a method of using a polynucleotide of any (I)-(XLVIII) or        peptide of (XLIX) or a composition of (L) in a method to        diagnose, prophylax, prognose, or treat a a cancer of a tissue        listed in Table I; and,    -   (LVI) a method of using a polynucleotide of any (I)-(XLVIII) or        peptide of (XLIX) or a composition of (L) in a method to        identify or characterize a modulator of a cell expressing        193P1E1B.

As used herein, a range is understood to disclose specifically all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 193P1E1Bpolynucleotides that encode specific portions of 193P1E1B mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375,400, 410, 412 or more contiguous amino acids of 193P1E1B variant 1; themaximal lengths relevant for other variants are: variant 5, 412 aminoacids; variant 6, 412 amino acids, variant 9, 330 amino acids, variant10, 388 amino acids, variant 11, 308 amino acids, variant 12, 73 aminoacids, and variant 13, 372 amino acids.

For example, representative embodiments of the invention disclosedherein include: polynucleotides and their encoded peptides themselvesencoding about amino acid 1 to about amino acid 10 of the 193P1E1Bprotein shown in FIG. 2 or FIG. 3, polynucleotides encoding about aminoacid 10 to about amino acid 20 of the 193P1E1B protein shown in FIG. 2or FIG. 3, polynucleotides encoding about amino acid 20 to about aminoacid 30 of the 193P1E1B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 30 to about amino acid 40 ofthe 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 40 to about amino acid 50 of the 193P1E1B protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 toabout amino acid 60 of the 193P1E1B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 60 to about amino acid 70 ofthe 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encodingabout amino acid 70 to about amino acid 80 of the 193P1E1B protein shownin FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 toabout amino acid 90 of the 193P1E1B protein shown in FIG. 2 or FIG. 3,polynucleotides encoding about amino acid 90 to about amino acid 100 ofthe 193P1E1B protein shown in FIG. 2 or FIG. 3, in increments of about10 amino acids, ending at the carboxyl terminal amino acid set forth inFIG. 2 or FIG. 3. Accordingly, polynucleotides encoding portions of theamino acid sequence (of about 10 amino acids), of amino acids, 100through the carboxyl terminal amino acid of the 193P1E1B protein areembodiments of the invention. Wherein it is understood that eachparticular amino acid position discloses that position plus or minusfive amino acid residues.

Polynucleotides encoding relatively long portions of a 193P1E1B proteinare also within the scope of the invention. For example, polynucleotidesencoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about aminoacid 20, (or 30, or 40 or 50 etc.) of the 193P1E1B protein “or variant”shown in FIG. 2 or FIG. 3 can be generated by a variety of techniqueswell known in the art. These polynucleotide fragments can include anyportion of the 193P1E1B sequence as shown in FIG. 2.

Additional illustrative embodiments of the invention disclosed hereininclude 193P1E1B polynucleotide fragments encoding one or more of thebiological motifs contained within a 193P1E1B protein “or variant”sequence, including one or more of the motif-bearing subsequences of a193P1E1B protein “or variant” set forth in Tables VIII-XXI andXXII-XLIX. In another embodiment, typical polynucleotide fragments ofthe invention encode one or more of the regions of 193P1E1B protein orvariant that exhibit homology to a known molecule. In another embodimentof the invention, typical polynucleotide fragments can encode one ormore of the 193P1E1B protein or variant N-glycosylation sites, cAMP andcGMP-dependent protein kinase phosphorylation sites, casein kinase IIphosphorylation sites or N-myristoylation site and amidation sites.

Note that to determine the starting position of any peptide set forth inTables VIII-XXI and Tables XXII to XLIX (collectively HLA PeptideTables) respective to its parental protein, e.g., variant 1, variant 2,etc., reference is made to three factors: the particular variant, thelength of the peptide in an HLA Peptide Table, and the Search Peptideslisted in Table LVII. Generally, a unique Search Peptide is used toobtain HLA peptides for a particular variant. The position of eachSearch Peptide relative to its respective parent molecule is listed inTable VII. Accordingly, if a Search Peptide begins at position “X”, onemust add the value “X minus 1” to each position in Tables VIII-XXI andTables XXII-IL to obtain the actual position of the HLA peptides intheir parental molecule. For example if a particular Search Peptidebegins at position 150 of its parental molecule, one must add 150 -1,i.e., 149 to each HLA peptide amino acid position to calculate theposition of that amino acid in the parent molecule.

II.A.) Uses of 193P1E1B Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number ofdifferent specific uses. The human 193P1E1B gene maps to the chromosomallocation set forth in the Example entitled “Chromosomal Mapping of193P1E1B.” For example, because the 193P1E1B gene maps to thischromosome, polynucleotides that encode different regions of the193P1E1B proteins are used to characterize cytogenetic abnormalities ofthis chromosomal locale, such as abnormalities that are identified asbeing associated with various cancers. In certain genes, a variety ofchromosomal abnormalities including rearrangements have been identifiedas frequent cytogenetic abnormalities in a number of different cancers(see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998);Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al.,P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encodingspecific regions of the 193P1E1B proteins provide new tools that can beused to delineate, with greater precision than previously possible,cytogenetic abnormalities in the chromosomal region that encodes193P1E1B that may contribute to the malignant phenotype. In thiscontext, these polynucleotides satisfy a need in the art for expandingthe sensitivity of chromosomal screening in order to identify moresubtle and less common chromosomal abnormalities (see e.g. Evans et al.,Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as 193P1E1B was shown to be highly expressed in bladder andother cancers, 193P1E1B polynucleotides are used in methods assessingthe status of 193P1E1B gene products in normal versus cancerous tissues.Typically, polynucleotides that encode specific regions of the 193P1E1Bproteins are used to assess the presence of perturbations (such asdeletions, insertions, point mutations, or alterations resulting in aloss of an antigen etc.) in specific regions of the 193P1E1B gene, suchas regions containing one or more motifs. Exemplary assays include bothRT-PCR assays as well as single-strand conformation polymorphism (SSCP)analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378(1999), both of which utilize polynucleotides encoding specific regionsof a protein to examine these regions within the protein.

II.A.2.) Antisense Embodiments

Other specifically contemplated nucleic acid related embodiments of theinvention disclosed herein are genomic DNA, cDNAs, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone, or including alternative bases, whether derivedfrom natural sources or synthesized, and include molecules capable ofinhibiting the RNA or protein expression of 193P1E1B. For example,antisense molecules can be RNAs or other molecules, including peptidenucleic acids (PNAs) or non-nucleic acid molecules such asphosphorothioate derivatives that specifically bind DNA or RNA in a basepair-dependent manner. A skilled artisan can readily obtain theseclasses of nucleic acid molecules using the 193P1E1B polynucleotides andpolynucleotide sequences disclosed herein.

Antisense technology entails the administration of exogenousoligonucleotides that bind to a target polynucleotide located within thecells. The term “antisense” refers to the fact that sucholigonucleotides are complementary to their intracellular targets, e.g.,193P1E1B. See for example, Jack Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5(1988). The 193P1E1B antisense oligonucleotides of the present inventioninclude derivatives such as S-oligonucleotides (phosphorothioatederivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhancedcancer cell growth inhibitory action. S-oligos (nucleosidephosphorothioates) are isoelectronic analogs of an oligonucleotide(O-oligo) in which a nonbridging oxygen atom of the phosphate group isreplaced by a sulfur atom. The S-oligos of the present invention can beprepared by treatment of the corresponding O-oligos with3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transferreagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254(1990).Additional 193P1E1B antisense oligonucleotides of the present inventioninclude morpholino antisense oligonucleotides known in the art (see,e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development6: 169-175).

The 193P1E1B antisense oligonucleotides of the present inventiontypically can be RNA or DNA that is complementary to and stablyhybridizes with the first 100 5′ codons or last 100 3′ codons of a193P1E1B genomic sequence or the corresponding mRNA. Absolutecomplementarity is not required, although high degrees ofcomplementarity are preferred. Use of an oligonucleotide complementaryto this region allows for the selective hybridization to 193P1E1B mRNAand not to mRNA specifying other regulatory subunits of protein kinase.In one embodiment, 193P1E1B antisense oligonucleotides of the presentinvention are 15 to 30-mer fragments of the antisense DNA molecule thathave a sequence that hybridizes to 193P1E1B mRNA. Optionally, 193P1E1Bantisense oligonucleotide is a 30-mer oligonucleotide that iscomplementary to a region in the first 10 5′ codons or last 10 3′ codonsof 193P1E1B. Alternatively, the antisense molecules are modified toemploy ribozymes in the inhibition of 193P1E1B expression, see, e.g., L.A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3.) Primers and Primer Pairs

Further specific embodiments of these nucleotides of the inventioninclude primers and primer pairs, which allow the specific amplificationof polynucleotides of the invention or of any specific parts thereof,and probes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes can be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers are used todetect the presence of a 193P1E1B polynucleotide in a sample and as ameans for detecting a cell expressing a 193P1E1B protein.

Examples of such probes include polypeptides comprising all or part ofthe human 193P1E1B cDNA sequence shown in FIG. 2. Examples of primerpairs capable of specifically amplifying 193P1E1B mRNAs are alsodescribed in the Examples. As will be understood by the skilled artisan,a great many different primers and probes can be prepared based on thesequences provided herein and used effectively to amplify and/or detecta 193P1E1B mRNA.

The 193P1E1B polynucleotides of the invention are useful for a varietyof purposes, including but not limited to their use as probes andprimers for the amplification and/or detection of the 193P1E1B gene(s),mRNA(s), or fragments thereof; as reagents for the diagnosis and/orprognosis of prostate cancer and other cancers; as coding sequencescapable of directing the expression of 193P1E1B polypeptides; as toolsfor modulating or inhibiting the expression of the 193P1E1B gene(s)and/or translation of the 193P1E1B transcript(s); and as therapeuticagents.

The present invention includes the use of any probe as described hereinto identify and isolate a 193P1E1B or 193P1E1B related nucleic acidsequence from a naturally occurring source, such as humans or othermammals, as well as the isolated nucleic acid sequence per se, whichwould comprise all or most of the sequences found in the probe used.

II.A.4.) Isolation of 193P1E1B-Encoding Nucleic Acid Molecules

The 193P1E1B cDNA sequences described herein enable the isolation ofother polynucleotides encoding 193P1E1B gene product(s), as well as theisolation of polynucleotides encoding 193P1E1B gene product homologs,alternatively spliced isoforms, allelic variants, and mutant forms of a193P1E1B gene product as well as polynucleotides that encode analogs of193P1E1B-related proteins. Various molecular cloning methods that can beemployed to isolate full length cDNAs encoding a 193P1E1B gene are wellknown (see, for example, Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989;Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley andSons, 1995). For example, lambda phage cloning methodologies can beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing 193P1E1Bgene cDNAs can be identified by probing with a labeled 193P1E1B cDNA ora fragment thereof. For example, in one embodiment, a 193P1E1B cDNA(e.g., FIG. 2) or a portion thereof can be synthesized and used as aprobe to retrieve overlapping and full-length cDNAs corresponding to a193P1E1B gene. A 193P1E1B gene itself can be isolated by screeninggenomic DNA libraries, bacterial artificial chromosome libraries (BACs),yeast artificial chromosome libraries (YACs), and the like, with193P1E1B DNA probes or primers.

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga 193P1E1B polynucleotide, a fragment, analog or homologue thereof,including but not limited to phages, plasmids, phagemids, cosmids, YACs,BACs, as well as various viral and non-viral vectors well known in theart and cells transformed or transfected with such recombinant DNA orRNA molecules. Methods for generating such molecules are well known(see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a 193P1E1B polynucleotide, fragment,analog or homologue thereof within a suitable prokaryotic or eukaryotichost cell. Examples of suitable eukaryotic host cells include a yeastcell, a plant cell, or an animal cell, such as a mammalian cell or aninsect cell (e.g., a baculovirus-infectible cell such as an Sf9 orHighFive cell). Examples of suitable mammalian cells include variousprostate cancer cell lines such as DU145 and TsuPrl, other transfectableor transducible prostate cancer cell lines, primary cells (PrEC), aswell as a number of mammalian cells routinely used for the expression ofrecombinant proteins (e.g., COS, CHO, 293, 293T cells). Moreparticularly, a polynucleotide comprising the coding sequence of193P1E1B or a fragment, analog or homolog thereof can be used togenerate 193P1E1B proteins or fragments thereof using any number ofhost-vector systems routinely used and widely known in the art.

A wide range of host-vector systems suitable for the expression of193P1E1B proteins or fragments thereof are available, see for example,Sambrook et al., 1989, supra; Current Protocols in Molecular Biology,1995, supra). Preferred vectors for mammalian expression include but arenot limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviralvector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using theseexpression vectors, 193P1E1B can be expressed in several prostate cancerand non-prostate cell lines, including for example 293, 293T, rat-1, NIH3T3 and TsuPrl. The host-vector systems of the invention are useful forthe production of a 193P1E1B protein or fragment thereof. Suchhost-vector systems can be employed to study the functional propertiesof 193P1E1B and 193P1E1B mutations or analogs.

Recombinant human 193P1E1B protein or an analog or homolog or fragmentthereof can be produced by mammalian cells transfected with a constructencoding a 193P1E1B-related nucleotide. For example, 293T cells can betransfected with an expression plasmid encoding 193P1E1B or fragment,analog or homolog thereof, a 193P1E1B-related protein is expressed inthe 293T cells, and the recombinant 193P1E1B protein is isolated usingstandard purification methods (e.g., affinity purification usinganti-193P1E1B antibodies). In another embodiment, a 193P1E1B codingsequence is subcloned into the retroviral vector pSRαMSVtkneo and usedto infect various mammalian cell lines, such as NIH 3T3, TsuPr1,293 andrat-1 in order to establish 193P1E1B expressing cell lines. Variousother expression systems well known in the art can also be employed.Expression constructs encoding a leader peptide joined in frame to a193P1E1B coding sequence can be used for the generation of a secretedform of recombinant 193P1E1B protein.

As discussed herein, redundancy in the genetic code permits variation in193P1E1B gene sequences. In particular, it is known in the art thatspecific host species often have specific codon preferences, and thusone can adapt the disclosed sequence as preferred for a desired host.For example, preferred analog codon sequences typically have rare codons(i.e., codons having a usage frequency of less than about 20% in knownsequences of the desired host) replaced with higher frequency codons.Codon preferences for a specific species are calculated, for example, byutilizing codon usage tables available on the INTERNET.

Additional sequence modifications are known to enhance proteinexpression in a cellular host. These include elimination of sequencesencoding spurious polyadenylation signals, exon/intron splice sitesignals, transposon-like repeats, and/or other such well-characterizedsequences that are deleterious to gene expression. The GC content of thesequence is adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Wherepossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures. Other useful modifications include the addition of atranslational initiation consensus sequence at the start of the openreading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080(1989). Skilled artisans understand that the general rule thateukaryotic ribosomes initiate translation exclusively at the 5′ proximalAUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) 193P1E1B-Related Proteins

Another aspect of the present invention provides 193P1E1B-relatedproteins. Specific embodiments of 193P1E1B proteins comprise apolypeptide having all or part of the amino acid sequence of human193P1E1B as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of193P1E1B proteins comprise variant, homolog or analog polypeptides thathave alterations in the amino acid sequence of 193P1E1B shown in FIG. 2or FIG. 3.

Embodiments of a 193P1E1B polypeptide include: a 193P1E1B polypeptidehaving a sequence shown in FIG. 2, a peptide sequence of a 193P1E1B asshown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of apolypeptide having the sequence as shown in FIG. 2; or, at least 10contiguous peptides of a polypeptide having the sequence as shown inFIG. 2 where T is U. For example, embodiments of 193P1E1B peptidescomprise, without limitation:

-   -   (I) a protein comprising, consisting essentially of, or        consisting of an amino acid sequence as shown in FIGS. 2A-M or        FIGS. 3A-H;    -   (II) a 193P1E1B-related protein that is at least 90, 91, 92, 93,        94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino        acid sequence shown in FIGS. 2A-M;    -   (III) a 193P1E1B-related protein that is at least 90, 91, 92,        93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino        acid sequence shown in FIGS. 2A-M or 3A-H;    -   (IV) a protein that comprises at least one peptide set forth in        Tables VIII to XLIX, optionally with a proviso that it is not an        entire protein of FIG. 2;    -   (V) a protein that comprises at least one peptide set forth in        Tables VIII-XXI, collectively, which peptide is also set forth        in Tables XXII to XLIX, collectively, optionally with a proviso        that it is not an entire protein of FIG. 2;    -   (VI) a protein that comprises at least two peptides selected        from the peptides set forth in Tables VIII-XLIX, optionally with        a proviso that it is not an entire protein of FIG. 2;    -   (VII) a protein that comprises at least two peptides selected        from the peptides set forth in Tables VIII to XLIX collectively,        with a proviso that the protein is not a contiguous sequence        from an amino acid sequence of FIG. 2;    -   (VIII) a protein that comprises at least one peptide selected        from the peptides set forth in Tables VIII-XXI; and at least one        peptide selected from the peptides set forth in Tables XXII to        XLIX, with a proviso that the protein is not a contiguous        sequence from an amino acid sequence of FIG. 2;    -   (IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment        up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value greater        than 0.5 in the Hydrophilicity profile of FIG. 5;    -   (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A,        3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up        to 412, 412, 412, 330, 388, 308, 73, or 372 respectively, that        includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,        15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,        31, 32, 33, 34, 35 amino acid position(s) having a value less        than 0.5 in the Hydropathicity profile of FIG. 6;    -   (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment        up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively,        that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Percent Accessible Residues profile of        FIG. 7;    -   (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment        up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively,        that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Average Flexibility profile of FIG. 8;    -   (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11,        12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,        28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS.        3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment        up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively,        that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,        29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value        greater than 0.5 in the Beta-turn profile of FIG. 9;    -   (XIV) a peptide that occurs at least twice in Tables VIII-XXI        and XXII to XLIX, collectively;    -   (XV) a peptide that occurs at least three times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVI) a peptide that occurs at least four times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVII) a peptide that occurs at least five times in Tables        VIII-XXI and XXII to XLIX, collectively;    -   (XVIII) a peptide that occurs at least once in Tables VIII-XXI,        and at least once in tables XXII to XLIX;    -   (XIX) a peptide that occurs at least once in Tables VIII-XXI,        and at least twice in tables XXII to XLIX;    -   (XX) a peptide that occurs at least twice in Tables VIII-XXI,        and at least once in tables XXII to XLIX;    -   (XXI) a peptide that occurs at least twice in Tables VIII-XXI,        and at least twice in tables XXII to XLIX;    -   (XXII) a peptide which comprises one two, three, four, or five        of the following characteristics, or an oligonucleotide encoding        such peptide:        -   i) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Hydrophilicity profile of FIG. 5;        -   ii) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or less than            0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in            the Hydropathicity profile of FIG. 6;        -   iii) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Percent Accessible Residues profile of FIG. 7;        -   iv) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Average Flexibility profile of FIG. 8; or,        -   v) a region of at least 5 amino acids of a particular            peptide of FIG. 3, in any whole number increment up to the            full length of that protein in FIG. 3, that includes an            amino acid position having a value equal to or greater than            0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in            the Beta-turn profile of FIG. 9;    -   (XXIII) a composition comprising a peptide of (I)-(XXII) or an        antibody or binding region thereof together with a        pharmaceutical excipient and/or in a human unit dose form.    -   (XXIV) a method of using a peptide of (I)-(XXII), or an antibody        or binding region thereof or a composition of (XXIII) in a        method to modulate a cell expressing 193P1E1B,    -   (XXV) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition of (XXIII) in a        method to diagnose, prophylax, prognose, or treat an individual        who bears a cell expressing 193P1E1B    -   (XXVI) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition (XXIII) in a method        to diagnose, prophylax, prognose, or treat an individual who        bears a cell expressing 193P1E1B, said cell from a cancer of a        tissue listed in Table I;    -   (XXVII) a method of using a peptide of (I)-(XXII) or an antibody        or binding region thereof or a composition of (XXIII) in a        method to diagnose, prophylax, prognose, or treat a a cancer;    -   (XXVIII) a method of using a peptide of (l)-(XXII) or an        antibody or binding region thereof or a composition of (XXIII)        in a method to diagnose, prophylax, prognose, or treat a a        cancer of a tissue listed in Table I; and,    -   (XXIX) a method of using a a peptide of (I)-(XXII) or an        antibody or binding region thereof or a composition (XXIII) in a        method to identify or characterize a modulator of a cell        expressing 193P1E1B.

As used herein, a range is understood to specifically disclose all wholeunit positions thereof.

Typical embodiments of the invention disclosed herein include 193P1E1Bpolynucleotides that encode specific portions of 193P1E1B mRNA sequences(and those which are complementary to such sequences) such as those thatencode the proteins and/or fragments thereof, for example:

(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375,400, 410, 412 or more contiguous amino acids of 193P1E1B variant 1; themaximal lengths relevant for other variants are: variant 5, 412 aminoacids; variant 6, 412 amino acids, variant 9, 330, variant 10, 388 aminoacids, variant 11, 308 amino acids, variant 12, 73, and variant 13, 372amino acids.

In general, naturally occurring allelic variants of human 193P1E1B sharea high degree of structural identity and homology (e.g., 90% or morehomology). Typically, allelic variants of a 193P1E1B protein containconservative amino acid substitutions within the 193P1E1B sequencesdescribed herein or contain a substitution of an amino acid from acorresponding position in a homologue of 193P1E1B. One class of 193P1E1Ballelic variants are proteins that share a high degree of homology withat least a small region of a particular 193P1E1B amino acid sequence,but further contain a radical departure from the sequence, such as anon-conservative substitution, truncation, insertion or frame shift. Incomparisons of protein sequences, the terms, similarity, identity, andhomology each have a distinct meaning as appreciated in the field ofgenetics. Moreover, orthology and paralogy can be important conceptsdescribing the relationship of members of a given protein family in oneorganism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative aminoacid substitutions can frequently be made in a protein without alteringeither the conformation or the function of the protein. Proteins of theinvention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15conservative substitutions. Such changes include substituting any ofisoleucine (I), valine (V), and leucine (L) for any other of thesehydrophobic amino acids; aspartic acid (D) for glutamic acid (E) andvice versa; glutamine (Q) for asparagine (N) and vice versa; and serine(S) for threonine (T) and vice versa. Other substitutions can also beconsidered conservative, depending on the environment of the particularamino acid and its role in the three-dimensional structure of theprotein. For example, glycine (G) and alanine (A) can frequently beinterchangeable, as can alanine (A) and valine (V). Methionine (M),which is relatively hydrophobic, can frequently be interchanged withleucine and isoleucine, and sometimes with valine. Lysine (K) andarginine (R) are frequently interchangeable in locations in which thesignificant feature of the amino acid residue is its charge and thediffering pK's of these two amino acid residues are not significant.Still other changes can be considered “conservative” in particularenvironments (see, e.g. Table III herein; pages 13-15 “Biochemistry”2^(nd) ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995;270(20):11882-6).

Embodiments of the invention disclosed herein include a wide variety ofart-accepted variants or analogs of 193P1E1B proteins such aspolypeptides having amino acid insertions, deletions and substitutions.193P1E1B variants can be made using methods known in the art such assite-directed mutagenesis, alanine scanning, and PCR mutagenesis.Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331(1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassettemutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selectionmutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415(1986)) or other known techniques can be performed on the cloned DNA toproduce the 193P1E1B variant DNA.

Scanning amino acid analysis can also be employed to identify one ormore amino acids along a contiguous sequence that is involved in aspecific biological activity such as a protein-protein interaction.Among the preferred scanning amino acids are relatively small, neutralamino acids. Such amino acids include alanine, glycine, serine, andcysteine. Alanine is typically a preferred scanning amino acid amongthis group because it eliminates the side-chain beyond the beta-carbonand is less likely to alter the main-chain conformation of the variant.Alanine is also typically preferred because it is the most common aminoacid. Further, it is frequently found in both buried and exposedpositions (Creighton, The Proteins, (W. H. Freeman & Co., N.Y.);Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does notyield adequate amounts of variant, an isosteric amino acid can be used.

As defined herein, 193P1E1B variants, analogs or homologs, have thedistinguishing attribute of having at least one epitope that is “crossreactive” with a 193P1E1B protein having an amino acid sequence of FIG.3. As used in this sentence, “cross reactive” means that an antibody orT cell that specifically binds to a 193P1E1B variant also specificallybinds to a 193P1E1B protein having an amino acid sequence set forth inFIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG.3, when it no longer contains any epitope capable of being recognized byan antibody or T cell that specifically binds to the starting 193P1E1Bprotein. Those skilled in the art understand that antibodies thatrecognize proteins bind to epitopes of varying size, and a grouping ofthe order of about four or five amino acids, contiguous or not, isregarded as a typical number of amino acids in a minimal epitope. See,e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al.,Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985)135(4):2598-608.

Other classes of 193P1E1B-related protein variants share 70%, 75%, 80%,85% or 90% or more similarity with an amino acid sequence of FIG. 3, ora fragment thereof. Another specific class of 193P1E1B protein variantsor analogs comprises one or more of the 193P1E1B biological motifsdescribed herein or presently known in the art. Thus, encompassed by thepresent invention are analogs of 193P1E1B fragments (nucleic or aminoacid) that have altered functional (e.g. immunogenic) propertiesrelative to the starting fragment. It is to be appreciated that motifsnow or which become part of the art are to be applied to the nucleic oramino acid sequences of FIG. 2 or FIG. 3.

As discussed herein, embodiments of the claimed invention includepolypeptides containing less than the full amino acid sequence of a193P1E1B protein shown in FIG. 2 or FIG. 3. For example, representativeembodiments of the invention comprise peptides/proteins having any 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a193P1E1B protein shown in FIG. 2 or FIG. 3.

Moreover, representative embodiments of the invention disclosed hereininclude polypeptides consisting of about amino acid 1 to about aminoacid 10 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptidesconsisting of about amino acid 10 to about amino acid 20 of a 193P1E1Bprotein shown in FIG. 2 or FIG. 3, polypeptides consisting of aboutamino acid 20 to about amino acid 30 of a 193P1E1B protein shown in FIG.2 or FIG. 3, polypeptides consisting of about amino acid 30 to aboutamino acid 40 of a 193P1E1B protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 40 to about amino acid 50 ofa 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 50 to about amino acid 60 of a 193P1E1B protein shownin FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 toabout amino acid 70 of a 193P1E1B protein shown in FIG. 2 or FIG. 3,polypeptides consisting of about amino acid 70 to about amino acid 80 ofa 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting ofabout amino acid 80 to about amino acid 90 of a 193P1E1B protein shownin FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 toabout amino acid 100 of a 193P1E1B protein shown in FIG. 2 or FIG. 3,etc. throughout the entirety of a 193P1E1B amino acid sequence.Moreover, polypeptides consisting of about amino acid 1 (or 20or 30 or40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a193P1E1B protein shown in FIG. 2 or FIG. 3 are embodiments of theinvention. It is to be appreciated that the starting and stoppingpositions in this paragraph refer to the specified position as well asthat position plus or minus 5 residues.

193P1E1B-related proteins are generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the art.Alternatively, recombinant methods can be used to generate nucleic acidmolecules that encode a 193P1E1B-related protein. In one embodiment,nucleic acid molecules provide a means to generate defined fragments ofa 193P1E1B protein (or variants, homologs or analogs thereof).

III.A.) Motif-Bearing Protein Embodiments

Additional illustrative embodiments of the invention disclosed hereininclude 193P1E1B polypeptides comprising the amino acid residues of oneor more of the biological motifs contained within a 193P1E1B polypeptidesequence set forth in FIG. 2 or FIG. 3. Various motifs are known in theart, and a protein can be evaluated for the presence of such motifs by anumber of publicly available Internet sites.

Motif bearing subsequences of all 193P1E1B variant proteins are setforth and identified in Tables VIII-XXI and XXII-XLIX.

Table V sets forth several frequently occurring motifs based on pfamsearches. The columns of Table V list (1) motif name abbreviation, (2)percent identity found amongst the different member of the motif family,(3) motif name or description and (4) most common function; locationinformation is included if the motif is relevant for location.

Polypeptides comprising one or more of the 193P1E1B motifs discussedabove are useful in elucidating the specific characteristics of amalignant phenotype in view of the observation that the 193P1E1B motifsdiscussed above are associated with growth dysregulation and because193P1E1B is overexpressed in certain cancers (See, e.g., Table I).Casein kinase 11, cAMP and camp-dependent protein kinase, and ProteinKinase C, for example, are enzymes known to be associated with thedevelopment of the malignant phenotype (see e.g. Chen et al., LabInvest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10):4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian,Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation andmyristoylation are protein modifications also associated with cancer andcancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154(1997)). Amidation is another protein modification also associated withcancer and cancer progression (see e.g. Treston et al., J. Natl. CancerInst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins of the invention comprise one or more ofthe immunoreactive epitopes identified in accordance with art-acceptedmethods, such as the peptides set forth in Tables VIII-XXI andXXII-XLIX. CTL epitopes can be determined using specific algorithms toidentify peptides within a 193P1E1B protein that are capable ofoptimally binding to specified HLA alleles. Moreover, processes foridentifying peptides that have sufficient binding affinity for HLAmolecules and which are correlated with being immunogenic epitopes, arewell known in the art, and are carried out without undueexperimentation. In addition, processes for identifying peptides thatare immunogenic epitopes, are well known in the art, and are carried outwithout undue experimentation either in vitro or in vivo.

Also known in the art are principles for creating analogs of suchepitopes in order to modulate immunogenicity. For example, one beginswith an epitope that bears a CTL or HTL motif (see, e.g., the HLA ClassI and HLA Class II motifs/supermotifs of Table IV). The epitope isanaloged by substituting out an amino acid at one of the specifiedpositions, and replacing it with another amino acid specified for thatposition. For example, on the basis of residues defined in Table IV, onecan substitute out a deleterious residue in favor of any other residue,such as a preferred residue; substitute a less-preferred residue with apreferred residue; or substitute an originally-occurring preferredresidue with another preferred residue. Substitutions can occur atprimary anchor positions or at other positions in a peptide; see, e.g.,Table IV.

A variety of references reflect the art regarding the identification andgeneration of epitopes in a protein of interest as well as analogsthereof. See, for example, WO 97/33602 to Chesnut et al.; Sette,Immunogenetics 1999 50(34): 201-212; Sette et al., J. Immunol. 2001166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondoet al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol.1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt etal., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7(1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3):266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J.Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9):751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments of the invention include polypeptides comprisingcombinations of the different motifs set forth in Table VI, and/or, oneor more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX,and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX,and/or, one or more of the T cell binding motifs known in the art.Preferred embodiments contain no insertions, deletions or substitutionseither within the motifs or within the intervening sequences of thepolypeptides. In addition, embodiments which include a number of eitherN-terminal and/or C-terminal amino acid residues on either side of thesemotifs may be desirable (to, for example, include a greater portion ofthe polypeptide architecture in which the motif is located). Typically,the number of N-terminal and/or C-terminal amino acid residues on eitherside of a motif is between about 1 to about 100 amino acid residues,preferably 5 to about 50 amino acid residues.

193P1E1B-related proteins are embodied in many forms, preferably inisolated form. A purified 193P1E1B protein molecule will besubstantially free of other proteins or molecules that impair thebinding of 193P1E1B to antibody, T cell or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a 193P1E1B-related proteins include purified193P1E1B-related proteins and functional, soluble 193P1E1B-relatedproteins. In one embodiment, a functional, soluble 193P1E1B protein orfragment thereof retains the ability to be bound by antibody, T cell orother ligand.

The invention also provides 193P1E1B proteins comprising biologicallyactive fragments of a 193P1E1B amino acid sequence shown in FIG. 2 orFIG. 3. Such proteins exhibit properties of the starting 193P1E1Bprotein, such as the ability to elicit the generation of antibodies thatspecifically bind an epitope associated with the starting 193P1E1Bprotein; to be bound by such antibodies; to elicit the activation of HTLor CTL; and/or, to be recognized by HTL or CTL that also specificallybind to the starting protein.

193P1E1B-related polypeptides that contain particularly interestingstructures can be predicted and/or identified using various analyticaltechniques well known in the art, including, for example, the methods ofChou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis, or based on immunogenicity. Fragments thatcontain such structures are particularly useful in generatingsubunit-specific anti-193P1E1B antibodies or T cells or in identifyingcellular factors that bind to 193P1E1B. For example, hydrophilicityprofiles can be generated, and immunogenic peptide fragments identified,using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol.157:105-132. Percent (%) Accessible Residues profiles can be generated,and immunogenic peptide fragments identified, using the method of JaninJ., 1979, Nature 277:491492. Average Flexibility profiles can begenerated, and immunogenic peptide fragments identified, using themethod of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. ProteinRes. 32:242-255. Beta-turn profiles can be generated, and immunogenicpeptide fragments identified, using the method of Deleage, G., Roux B.,1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identifypeptides within a 193P1E1B protein that are capable of optimally bindingto specified HLA alleles (e.g., by using the SYFPEITHI site at WorldWide Web; Epimatrix™ and Epimer™, Brown University, URL and BIMAS.Illustrating this, peptide epitopes from 193P1E1B that are presented inthe context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11,A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX).Specifically, the complete amino acid sequence of the 193P1E1B proteinand relevant portions of other variants, i.e., for HLA Class Ipredictions 9 flanking residues on either side of a point mutation orexon junction, and for HLA Class II predictions 14 flanking residues oneither side of a point mutation or exon junction corresponding to thatvariant, were entered into the HLA Peptide Motif Search algorithm foundin the Bioinformatics and Molecular Analysis Section (BIMAS) web sitelisted above; in addition to the site SYFPEITHI.

The HLA peptide motif search algorithm was developed by Dr. Ken Parkerbased on binding of specific peptide sequences in the groove of HLAClass I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al,J. Immunol. 149:3580-7 (1992); Parker et at, J. Immunol. 152:163-75(1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and10-mer peptides from a complete protein sequence for predicted bindingto HLA-A2 as well as numerous other HLA Class I molecules. Many HLAclass I binding peptides are 8-, 9-, 10 or 11-mers. For example, forClass I HLA-A2, the epitopes preferably contain a leucine (L) ormethionine (M) at position 2 and a valine (V) or leucine (L) at theC-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)).Selected results of 193P1E1B predicted binding peptides are shown inTables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII,selected candidates, 9-mers and 10-mers, for each family member areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. In Tables XLVI-XLIX,selected candidates, 15-mers, for each family member are shown alongwith their location, the amino acid sequence of each specific peptide,and an estimated binding score. The binding score corresponds to theestimated half time of dissociation of complexes containing the peptideat 37° C. at pH 6.5. Peptides with the highest binding score arepredicted to be the most tightly bound to HLA Class I on the cellsurface for the greatest period of time and thus represent the bestimmunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated bystabilization of HLA expression on the antigen-processing defective cellline T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa etal., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides canbe evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes(CTL) in the presence of antigen presenting cells such as dendriticcells.

It is to be appreciated that every epitope predicted by the BIMAS site,Epimer™ and Epimatrix™ sites, or specified by the HLA class I or classII motifs available in the art or which become part of the art such asset forth in Table IV (or determined using SYFPETHI or BIMAS, are to be“applied” to a 193P1E1B protein in accordance with the invention. Asused in this context “applied” means that a 193P1E1B protein isevaluated, e.g., visually or by computer-based patterns finding methods,as appreciated by those of skill in the relevant art. Every subsequenceof a 193P1E1B protein of 8, 9, 10, or 11 amino acid residues that bearsan HLA Class I motif, or a subsequence of 9 or more amino acid residuesthat bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of 193P1E1B-related Proteins

In an embodiment described in the examples that follow, 193P1E1B can beconveniently expressed in cells (such as 293T cells) transfected with acommercially available expression vector such as a CMV-driven expressionvector encoding 193P1E1B with a C-terminal 6×His and MYC tag(pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, NashvilleTenn.). The Tag5 vector provides an IgGK secretion signal that can beused to facilitate the production of a secreted 193P1E1B protein intransfected cells. The secreted HIS-tagged 193P1E1B in the culture mediacan be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of 193P1E1B-Related Proteins

Modifications of 193P1E1B-related proteins such as covalentmodifications are included within the scope of this invention. One typeof covalent modification includes reacting targeted amino acid residuesof a 193P1E1B polypeptide with an organic derivatizing agent that iscapable of reacting with selected side chains or the N- or C-terminalresidues of a 193P1E1B protein. Another type of covalent modification ofa 193P1E1B polypeptide included within the scope of this inventioncomprises altering the native glycosylation pattern of a protein of theinvention. Another type of covalent modification of 193P1E1B compriseslinking a 193P1E1B polypeptide to one of a variety of nonproteinaceouspolymers, e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The 193P1E1B-related proteins of the present invention can also bemodified to form a chimeric molecule comprising 193P1E1B fused toanother, heterologous polypeptide or amino acid sequence. Such achimeric molecule can be synthesized chemically or recombinantly. Achimeric molecule can have a protein of the invention fused to anothertumor-associated antigen or fragment thereof. Alternatively, a proteinin accordance with the invention can comprise a fusion of fragments of a193P1E1B sequence (amino or nucleic acid) such that a molecule iscreated that is not, through its length, directly homologous to theamino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such achimeric molecule can comprise multiples of the same subsequence of193P1E1B. A chimeric molecule can comprise a fusion of a193P1E1B-related protein with a polyhistidine epitope tag, whichprovides an epitope to which immobilized nickel can selectively bind,with cytokines or with growth factors. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of a 193P1E1B protein. In analternative embodiment, the chimeric molecule can comprise a fusion of a193P1E1B-related protein with an immunoglobulin or a particular regionof an immunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The Ig fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a 193P1E1B polypeptide in place of at least one variable regionwithin an Ig molecule. In a preferred embodiment, the immunoglobulinfusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3regions of an IgGI molecule. For the production of immunoglobulinfusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.

III.D.) Uses of 193P1E1B-Related Proteins

The proteins of the invention have a number of different specific uses.As 193P1E1B is highly expressed in prostate and other cancers,193P1E1B-related proteins are used in methods that assess the status of193P1E1B gene products in normal versus cancerous tissues, therebyelucidating the malignant phenotype. Typically, polypeptides fromspecific regions of a 193P1E1B protein are used to assess the presenceof perturbations (such as deletions, insertions, point mutations etc.)in those regions (such as regions containing one or more motifs).Exemplary assays utilize antibodies or T cells targeting193P1E1B-related proteins comprising the amino acid residues of one ormore of the biological motifs contained within a 193P1E1B polypeptidesequence in order to evaluate the characteristics of this region innormal versus cancerous tissues or to elicit an immune response to theepitope. Alternatively, 193P1E1B-related proteins that contain the aminoacid residues of one or more of the biological motifs in a 193P1E1Bprotein are used to screen for factors that interact with that region of193P1E1B.

193P1E1B protein fragments/subsequences are particularly useful ingenerating and characterizing domain-specific antibodies (e.g.,antibodies recognizing an extracellular or intracellular epitope of a193P1E1B protein), for identifying agents or cellular factors that bindto 193P1E1B or a particular structural domain thereof, and in varioustherapeutic and diagnostic contexts, including but not limited todiagnostic assays, cancer vaccines and methods of preparing suchvaccines.

Proteins encoded by the 193P1E1B genes, or by analogs, homologs orfragments thereof, have a variety of uses, including but not limited togenerating antibodies and in methods for identifying ligands and otheragents and cellular constituents that bind to a 193P1E1B gene product.Antibodies raised against a 193P1E1B protein or fragment thereof areuseful in diagnostic and prognostic assays, and imaging methodologies inthe management of human cancers characterized by expression of 193P1E1Bprotein, such as those listed in Table I. Such antibodies can beexpressed intracellularly and used in methods of treating patients withsuch cancers. 193P1E1B-related nucleic acids or proteins are also usedin generating HTL or CTL responses.

Various immunological assays useful for the detection of 193P1E1Bproteins are used, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Antibodies can be labeled and used asimmunological imaging reagents capable of detecting 193P1E1B-expressingcells (e.g., in radioscintigraphic imaging methods). 193P1E1B proteinsare also particularly useful in generating cancer vaccines, as furtherdescribed herein.

IV.) 193P1E1B Antibodies

Another aspect of the invention provides antibodies that bind to193P1E1B-related proteins. Preferred antibodies specifically bind to a193P1E1B-related protein and do not bind (or bind weakly) to peptides orproteins that are not 193P1E1B-related proteins under physiologicalconditions. In this context, examples of physiological conditionsinclude: 1) phosphate buffered saline; 2) Tris-buffered salinecontaining 25mM Tris and 150 mM NaCl; or saline (0.9% NaCl); 4) animalserum such as human serum; or 5) a combination of any of 1) through 4);these reactions preferably taking place at pH 7.5, alternatively in arange of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5;also, these reactions taking place at a temperature between 4° C. to 37°C. For example, antibodies that bind 193P1E1B can bind 193P1E1B-relatedproteins such as the homologs or analogs thereof.

193P1E1B antibodies of the invention are particularly useful in cancer(see, e.g., Table I) diagnostic and prognostic assays, and imagingmethodologies. Similarly, such antibodies are useful in the treatment,diagnosis, and/or prognosis of other cancers, to the extent 193P1E1B isalso expressed or overexpressed in these other cancers. Moreover,intracellularly expressed antibodies (e.g., single chain antibodies) aretherapeutically useful in treating cancers in which the expression of193P1E1B is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for thedetection and quantification of 193P1E1B and mutant 193P1E1B-relatedproteins. Such assays can comprise one or more 193P1E1B antibodiescapable of recognizing and binding a 193P1E1B-related protein, asappropriate. These assays are performed within various immunologicalassay formats well known in the art, including but not limited tovarious types of radioimmunoassays, enzyme-linked immunosorbent assays(ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cellimmunogenicity assays (inhibitory or stimulatory) as well as majorhistocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostatecancer and other cancers expressing 193P1E1B are also provided by theinvention, including but not limited to radioscintigraphic imagingmethods using labeled 193P1E1B antibodies. Such assays are clinicallyuseful in the detection, monitoring, and prognosis of 193P1E1Bexpressing cancers such as prostate cancer.

193P1E1B antibodies are also used in methods for purifying a193P1E1B-related protein and for isolating 193P1E1B homologues andrelated molecules. For example, a method of purifying a 193P1E1B-relatedprotein comprises incubating a 193P1E1B antibody, which has been coupledto a solid matrix, with a lysate or other solution containing a193P1E1B-related protein under conditions that permit the 193P1E1Bantibody to bind to the 193P1E1B-related protein; washing the solidmatrix to eliminate impurities; and eluting the 193P1E1B-related proteinfrom the coupled antibody. Other uses of 193P1E1B antibodies inaccordance with the invention include generating ant-idiotypicantibodies that mimic a 193P1E1B protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies can be prepared by immunizing a suitablemammalian host using a 193P1E1B-related protein, peptide, or fragment,in isolated or immunoconjugated form (Antibodies: A Laboratory Manual,CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, ColdSpring Harbor Press, N.Y. (1989)). In addition, fusion proteins of193P1E1B can also be used, such as a 193P1E1B GST-fusion protein. In aparticular embodiment, a GST fusion protein comprising all or most ofthe amino add sequence of FIG. 2 or FIG. 3 is produced, then used as animmunogen to generate appropriate antibodies. In another embodiment, a193P1E1B-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used(with or without purified 193P1E1B-related protein or 193P1E1Bexpressing cells) to generate an immune response to the encodedimmunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648).

The amino acid sequence of a 193P1E1B protein as shown in FIG. 2 or FIG.3 can be analyzed to select specific regions of the 193P1E1B protein forgenerating antibodies. For example, hydrophobicity and hydrophilicityanalyses of a 193P1E1B amino acid sequence are used to identifyhydrophilic regions in the 193P1E1B structure. Regions of a 193P1E1Bprotein that show immunogenic structure, as well as other regions anddomains, can readily be identified using various other methods known inthe art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg,Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can begenerated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc.Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can begenerated using the method of Kyte, J. and Doolittle, R. F., 1982, J.Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can begenerated using the method of Janin J., 1979, Nature 277:491-492.Average Flexibility profiles can be generated-using the method ofBhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method ofDeleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, eachregion identified by any of these programs or methods is within thescope of the present invention. Methods for the generation of 193P1E1Bantibodies are further illustrated by way of the examples providedherein. Methods for preparing a protein or polypeptide for use as animmunogen are well known in the art. Also well known in the art aremethods for preparing immunogenic conjugates of a protein with acarrier, such as BSA, KLH or other carrier protein. In somecircumstances, direct conjugation using, for example, carbodiimidereagents are used; in other instances linking reagents such as thosesupplied by Pierce Chemical Co., Rockford, Ill., are effective.Administration of a 193P1E1B immunogen is often conducted by injectionover a suitable time period and with use of a suitable adjuvant, as isunderstood in the art During the immunization schedule, titers ofantibodies can be taken to determine adequacy of antibody formation.

193P1E1B monoclonal antibodies can be produced by various means wellknown in the art For example, immortalized cell lines that secrete adesired monoclonal antibody are prepared using the standard hybridomatechnology of Kohler and Milstein or modifications that immortalizeantibody-producing B cells, as is generally known. Immortalized celllines that secrete the desired antibodies are screened by immunoassay inwhich the antigen is a 193P1E1B-related protein. When the appropriateimmortalized cell culture is identified, the cells can be expanded andantibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, byrecombinant means. Regions that bind specifically to the desired regionsof a 193P1E1B protein can also be produced in the context of chimeric orcomplementarity-determining region (CDR) grafted antibodies of multiplespecies origin. Humanized or human 193P1E1B antibodies can also beproduced, and are preferred for use in therapeutic contexts. Methods forhumanizing murine and other non-human antibodies, by substituting one ormore of the non-human antibody CDRs for corresponding human antibodysequences, are well known (see for example, Jones et al., 1986, Nature321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen etal., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc.Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151:2296.

Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan et al., 1998,Nature Biotechnology 16: 535-539). Fully human 193P1E1B monoclonalantibodies can be generated using cloning technologies employing largehuman Ig gene combinatorial libraries (i.e., phage display) (Griffithsand Hoogenboom, Building an in vitro immune system: human antibodiesfrom phage display libraries. In: Protein Engineering of AntibodyMolecules for Prophylactic and Therapeutic Applications in Man, Clark,M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, HumanAntibodies from combinatorial libraries. Id., pp 65-82). Fully human193P1E1B monoclonal antibodies can also be produced using transgenicmice engineered to contain human immunoglobulin gene loci as describedin PCT Patent Application WO98/24893, Kuchelapati and Jakobovits et at,published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest.Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued Dec. 19, 2000;6,150,584 issued Nov. 12, 2000; and, 6,114,598 issued Sep. 5, 2000).This method avoids the in vitro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of 193P1E1B antibodies with a 193P1E1B-related protein can beestablished by a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,193P1E1B-related proteins, 193P1E1B-expressing cells or extractsthereof. A 193P1E1B antibody or fragment thereof can be labeled with adetectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more 193P1E1B epitopes are generated using methodsgenerally known in the art. Homodimeric antibodies can also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

V.) 193Pi E1B Cellular Immune Responses

The mechanism by which T cells recognize antigens has been delineated.Efficacious peptide epitope vaccine compositions of the invention inducea therapeutic or prophylactic immune responses in very broad segments ofthe world-wide population. For an understanding of the value andefficacy of compositions of the invention that induce cellular immuneresponses, a brief review of immunology-related technology is provided.

A complex of an HLA molecule and a peptidic antigen acts as the ligandrecognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071,1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. andBodmer, H., Annu. Rev. Immunol 7:601, 1989; Germain, R. N., Annu. Rev.Immunol. 11:403, 1993). Through the study of single amino acidsubstituted antigen analogs and the sequencing of endogenously bound,naturally processed peptides, critical residues that correspond tomotifs required for specific binding to HLA antigen molecules have beenidentified and are set forth in Table IV (see also, e.g., Southwood, etal., J. Immunol 160:3363, 1998; Rammensee, et al., Immunogenetics41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web;Sette, A. and Sidney, J. Curr. Opin. Immunol 10:478, 1998; Engelhard, V.H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr.Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol.6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J.Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol 157:3480-3490,1996; Sidney et al., Human Immunol. 45:79-93, 1996; Selle, A. andSidney, J. Immunogenetics 1999 Nov; 50(3-4):201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexeshave revealed pockets within the peptide binding cleft/groove of HLAmolecules which accommodate, in an allele-specific mode, residues borneby peptide ligands; these residues in turn determine the HLA bindingcapacity of the peptides in which they are present. (See, e.g., Madden,D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203,1996; Fremont et at, Immunity 8:305, 1998; Stern et al., Structure2:245, 1994; Jones, E. Y. Curr. Opin. Immunol 9:75, 1997; Brown, J. H.et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci.USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M.L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927,1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. MolBiol. 219:277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLAbinding motifs, or class I or class II supermotifs allows identificationof regions within a protein that are correlated with binding toparticular HLA antigen(s).

Thus, by a process of HLA motif identification, candidates forepitope-based vaccines have been identified; such candidates can befurther evaluated by HLA-peptide binding assays to determine bindingaffinity and/or the time period of association of the epitope and itscorresponding HLA molecule. Additional confirmatory work can beperformed to select, amongst these vaccine candidates, epitopes withpreferred characteristics in terms of population coverage, and/orimmunogenicity.

Various strategies can be utilized to evaluate cellular immunogenicity,including:

1) Evaluation of primary T cell cultures from normal individuals (see,e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. etal., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J.Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1,1998). This procedure involves the stimulation of peripheral bloodlymphocytes (PBL) from normal subjects with a test peptide in thepresence of antigen presenting cells in vitro over a period of severalweeks. T cells specific for the peptide become activated during thistime and are detected using, e.g., a lymphokine- or ⁵¹Cr-release assayinvolving peptide sensitized target cells.

2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. etal., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). Forexample, in such methods peptides in incomplete Freund's adjuvant areadministered subcutaneously to HLA transgenic mice. Several weeksfollowing immunization, splenocytes are removed and cultured in vitro inthe presence of test peptide for approximately one week.Peptide-specific T cells are detected using, e.g., a ⁵¹ Cr-release assayinvolving peptide sensitized target cells and target cells expressingendogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals whohave been either effectively vaccinated and/or from chronically illpatients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995;Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin.Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648,1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly,recall responses are detected by culturing PBL from subjects that havebeen exposed to the antigen due to disease and thus have generated animmune response “naturally”, or from patients who were vaccinatedagainst the antigen. PBL from subjects are cultured in vitro for 1-2weeks in the presence of test peptide plus antigen presenting cells(APC) to allow activation of “memory” T cells, as compared to “naive” Tcells. At the end of the culture period, T cell activity is detectedusing assays including ⁵¹ Cr release involving peptide-sensitizedtargets, T cell proliferation, or lymphokine release.

VI.) 193P1E1B Transgenic Animals

Nucleic acids that encode a 193P1E1B-related protein can also be used togenerate either transgenic animals or “knock out” animals that, in turn,are useful in the development and screening of therapeutically usefulreagents. In accordance with established techniques, cDNA encoding193P1E1B can be used to clone genomic DNA that encodes 193P1E1B. Thecloned genomic sequences can then be used to generate transgenic animalscontaining cells that express DNA that encode 193P1E1B. Methods forgenerating transgenic animals, particularly animals such as mice orrats, have become conventional in the art and are described, forexample, in U.S. Pat. Nos. 4,736,866 issued Apr. 12, 1988, and 4,870,009issued Sep. 26, 1989. Typically, particular cells would be targeted for193P1E1B transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 193P1E1Bcan be used to examine the effect of increased expression of DNA thatencodes 193P1E1B. Such animals can be used as tester animals forreagents thought to confer protection from, for example, pathologicalconditions associated with its overexpression. In accordance with thisaspect of the invention, an animal is treated with a reagent and areduced incidence of a pathological condition, compared to untreatedanimals that bear the transgene, would indicate a potential therapeuticintervention for the pathological condition.

Alternatively, non-human homologues of 193P1E1B can be used to constructa 193P1E1B “knockout” animal that has a defective or altered geneencoding 193P1E1B as a result of homologous recombination between theendogenous gene encoding 193P1E1B and altered genomic DNA encoding193P1E1B introduced into an embryonic cell of the animal. For example,cDNA that encodes 193P1E1B can be used to clone genomic DNA encoding193P1E1B in accordance with established techniques. A portion of thegenomic DNA encoding 193P1E1B can be deleted or replaced with anothergene, such as a gene encoding a selectable marker that can be used tomonitor integration. Typically, several kilobases of unaltered flankingDNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g.,Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedDNA has homologously recombined with the endogenous DNA are selected(see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are theninjected into a blastocyst of an animal (e.g., a mouse or rat) to formaggregation chimeras (see, e.g., Bradley, in Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL,Oxford, 1987), pp. 113-152). A chimeric embryo can then be implantedinto a suitable pseudopregnant female foster animal, and the embryobrought to term to create a “knock out” animal. Progeny harboring thehomologously recombined DNA in their germ cells can be identified bystandard techniques and used to breed animals in which all cells of theanimal contain the homologously recombined DNA. Knock out animals can becharacterized, for example, for their ability to defend against certainpathological conditions or for their development of pathologicalconditions due to absence of a 193P1E1B polypeptide.

VIII.) Methods for the Detection of 193P1E1B

Another aspect of the present invention relates to methods for detecting193P1E1B polynucleotides and 193P1E1B-related proteins, as well asmethods for identifying a cell that expresses 193P1E1B. The expressionprofile of 193P1E1B makes it a diagnostic marker for metastasizeddisease. Accordingly, the status of 193P1E1B gene products providesinformation useful for predicting a variety of factors includingsusceptibility to advanced stage disease, rate of progression, and/ortumor aggressiveness. As discussed in detail herein, the status of193P1E1B gene products in patient samples can be analyzed by a varietyprotocols that are well known in the art including immunohistochemicalanalysis, the variety of Northern blotting techniques including in situhybridization, RT-PCR analysis (for example on laser capturemicro-dissected samples), Western blot analysis and tissue arrayanalysis.

More particularly, the invention provides assays for the detection of193P1E1B polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable 193P1E1B polynucleotides include, for example, a193P1E1B gene or fragment thereof, 193P1E1B mRNA, alternative splicevariant 193P1E1B mRNAs, and recombinant DNA or RNA molecules thatcontain a 193P1E1B polynucleotide. A number of methods for amplifyingand/or detecting the presence of 193P1E1B polynucleotides are well knownin the art and can be employed in the practice of this aspect of theinvention.

In one embodiment, a method for detecting a 193P1E1B mRNA in abiological sample comprises producing cDNA from the sample by reversetranscription using at least one primer; amplifying the cDNA so producedusing a 193P1E1B polynucleotides as sense and antisense primers toamplify 193P1E1B cDNAs therein; and detecting the presence of theamplified 193P1E1B cDNA. Optionally, the sequence of the amplified193P1E1B cDNA can be determined.

In another embodiment, a method of detecting a 193P1E1B gene in abiological sample comprises first isolating genomic DNA from the sample;amplifying the isolated genomic DNA using 193P1E1B polynucleotides assense and antisense primers; and detecting the presence of the amplified193P1E1B gene. Any number of appropriate sense and antisense probecombinations can be designed from a 193P1E1B nucleotide sequence (see,e.g., FIG. 2) and used for this purpose.

The invention also provides assays for detecting the presence of a193P1E1B protein in a tissue or other biological sample such as serum,semen, bone, prostate, urine, cell preparations, and the like. Methodsfor detecting a 193P1E1B-related protein are also well known andinclude, for example, immunoprecipitation, immunohistochemical analysis,Western blot analysis, molecular binding assays, ELISA, ELIFA and thelike. For example, a method of detecting the presence of a193P1E1B-related protein in a biological sample comprises firstcontacting the sample with a 193P1E1B antibody, a 193P1E1B-reactivefragment thereof, or a recombinant protein containing an antigen-bindingregion of a 193P1E1B antibody; and then detecting the binding of193P1E1B-related protein in the sample.

Methods for identifying a cell that expresses 193P1E1B are also withinthe scope of the invention. In one embodiment, an assay for identifyinga cell that expresses a 193P1E1B gene comprises detecting the presenceof 193P1E1B mRNA in the cell. Methods for the detection of particularmRNAs in cells are well known and include, for example, hybridizationassays using complementary DNA probes (such as in situ hybridizationusing labeled 193P1E1B riboprobes, Northern blot and related techniques)and various nucleic acid amplification assays (such as RT-PCR usingcomplementary primers specific for 193P1E1B, and other amplificationtype detection methods, such as, for example, branched DNA, SISBA, TMAand the like). Alternatively, an assay for identifying a cell thatexpresses a 193P1E1B gene comprises detecting the presence of193P1E1B-related protein in the cell or secreted by the cell. Variousmethods for the detection of proteins are well known in the art and areemployed for the detection of 193P1E1B-related proteins and cells thatexpress 193P1E1B-related proteins.

193P1E1B expression analysis is also useful as a tool for identifyingand evaluating agents that modulate 193P1E1B gene expression. Forexample, 193P1E1B expression is significantly upregulated in prostatecancer, and is expressed in cancers of the tissues listed in Table I.Identification of a molecule or biological agent that inhibits 193P1E1Bexpression or over-expression in cancer cells is of therapeutic value.For example, such an agent can be identified by using a screen thatquantifies 193P1E1B expression by RT-PCR, nucleic acid hybridization orantibody binding.

VIII.) Methods for Monitoring the Status of 193P1E1B-Related Genes andTheir Products

Oncogenesis is known to be a multistep process where cellular growthbecomes progressively dysregulated and cells progress from a normalphysiological state to precancerous and then cancerous states (see,e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al.,Cancer Surv. 23: 19-32 (1995)). In this context, examining a biologicalsample for evidence of dysregulated cell growth (such as aberrant193P1E1B expression in cancers) allows for early detection of suchaberrant physiology, before a pathologic state such as cancer hasprogressed to a stage that therapeutic options are more limited and orthe prognosis is worse. In such examinations, the status of 193P1E1B ina biological sample of interest can be compared, for example, to thestatus of 193P1E1B in a corresponding normal sample (e.g. a sample fromthat individual or alternatively another individual that is not affectedby a pathology). An alteration in the status of 193P1E1B in thebiological sample (as compared to the normal sample) provides evidenceof dysregulated cellular growth. In addition to using a biologicalsample that is not affected by a pathology as a normal sample, one canalso use a predetermined normative value such as a predetermined normallevel of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol.Dec. 9, 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare193P1E1B status in a sample.

The term “status” in this context is used according to its art acceptedmeaning and refers to the condition or state of a gene and its products.Typically, skilled artisans use a number of parameters to evaluate thecondition or state of a gene and its products. These include, but arenot limited to the location of expressed gene products (including thelocation of 193P1E1B expressing cells) as well as the level, andbiological activity of expressed gene products (such as 193P1E1B mRNA,polynucleotides and polypeptides). Typically, an alteration in thestatus of 193P1E1B comprises a change in the location of 193P1E1B and/or193P1E1B expressing cells and/or an increase in 193P1E1B mRNA and/orprotein expression.

193P1E1B status in a sample can be analyzed by a number of means wellknown in the art, including without limitation, immunohistochemicalanalysis, in situ hybridization, RT-PCR analysis on laser capturemicro-dissected samples, Western blot analysis, and tissue arrayanalysis. Typical protocols for evaluating the status of a 193P1E1B geneand gene products are found, for example in Ausubel et al. eds., 1995,Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4(Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus,the status of 193P1E1B in a biological sample is evaluated by variousmethods utilized by skilled artisans including, but not limited togenomic Southern analysis (to examine, for example perturbations in a193P1E1B gene), Northern analysis and/or PCR analysis of 193P1E1B mRNA(to examine, for example alterations in the polynucleotide sequences orexpression levels of 193P1E1B mRNAs), and, Western and/orimmunohistochemical analysis (to examine, for example alterations inpolypeptide sequences, alterations in polypeptide localization within asample, alterations in expression levels of 193P1E1B proteins and/orassociations of 193P1E1B proteins with polypeptide binding partners).Detectable 193P1E1B polynucleotides include, for example, a 193P1E1Bgene or fragment thereof, 193P1E1B mRNA, alternative splice variants,193P1E1B mRNAs, and recombinant DNA or RNA molecules containing a193P1E1B polynucleotide.

The expression profile of 193P1E1B makes it a diagnostic marker forlocal and/or metastasized disease, and provides information on thegrowth or oncogenic potential of a biological sample. In particular, thestatus of 193P1E1B provides information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining 193P1E1B status and diagnosing cancers that express193P1E1B, such as cancers of the tissues listed in Table I. For example,because 193P1E1B mRNA is so highly expressed in prostate and othercancers relative to normal prostate tissue, assays that evaluate thelevels of 193P1E1B mRNA transcripts or proteins in a biological samplecan be used to diagnose a disease associated with 193P1E1Bdysregulation, and can provide prognostic information useful in definingappropriate therapeutic options.

The expression status of 193P1E1B provides information including thepresence, stage and location of dysplastic, precancerous and cancerouscells, predicting susceptibility to various stages of disease, and/orfor gauging tumor aggressiveness. Moreover, the expression profile makesit useful as an imaging reagent for metastasized disease. Consequently,an aspect of the invention is directed to the various molecularprognostic and diagnostic methods for examining the status of 193P1E1Bin biological samples such as those from individuals suffering from, orsuspected of suffering from a pathology characterized by dysregulatedcellular growth, such as cancer.

As described above, the status of 193P1E1B in a biological sample can beexamined by a number of well-known procedures in the art. For example,the status of 193P1E1B in a biological sample taken from a specificlocation in the body can be examined by evaluating the sample for thepresence or absence of 193P1E1B expressing cells (e.g. those thatexpress 193P1E1B mRNAs or proteins). This examination can provideevidence of dysregulated cellular growth, for example, when193P1E1B-expressing cells are found in a biological sample that does notnormally contain such cells (such as a lymph node), because suchalterations in the status of 193P1E1B in a biological sample are oftenassociated with dysregulated cellular growth. Specifically, oneindicator of dysregulated cellular growth is the metastases of cancercells from an organ of origin (such as the prostate) to a different areaof the body (such as a lymph node). In this context, evidence ofdysregulated cellular growth is important for example because occultlymph node metastases can be detected in a substantial proportion ofpatients with prostate cancer, and such metastases are associated withknown predictors of disease progression (see, e.g., Murphy et al.,Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1):17-28 (2000) and Freeman et al., J Urol August 1995 154(2 Pt 1):474-8).

In one aspect, the invention provides methods for monitoring 193P1E1Bgene products by determining the status of 193P1E1B gene productsexpressed by cells from an individual suspected of having a diseaseassociated with dysregulated cell growth (such as hyperplasia or cancer)and then comparing the status so determined to the status of 193P1E1Bgene products in a corresponding normal sample. The presence of aberrant193P1E1B gene products in the test sample relative to the normal sampleprovides an indication of the presence of dysregulated cell growthwithin the cells of the individual.

In another aspect, the invention provides assays useful in determiningthe presence of cancer in an individual, comprising detecting asignificant increase in 193P1E1B mRNA or protein expression in a testcell or tissue sample relative to expression levels in the correspondingnormal cell or tissue. The presence of 193P1E1B mRNA can, for example,be evaluated in tissues including but not limited to those listed inTable I. The presence of significant 193P1E1B expression in any of thesetissues is useful to indicate the emergence, presence and/or severity ofa cancer, since the corresponding normal tissues do not express 193P1E1BmRNA or express it at lower levels.

In a related embodiment, 193P1E1B status is determined at the proteinlevel rather than at the nucleic acid level. For example, such a methodcomprises determining the level of 193P1E1B protein expressed by cellsin a test tissue sample and comparing the level so determined to thelevel of 193P1E1B expressed in a corresponding normal sample. In oneembodiment, the presence of 193P1E1B protein is evaluated, for example,using immunohistochemical methods. 193P1E1B antibodies or bindingpartners capable of detecting 193P1E1B protein expression are used in avariety of assay formats well known in the art for this purpose.

In a further embodiment, one can evaluate the status of 193P1E1Bnucleotide and amino add sequences in a biological sample in order toidentify perturbations in the structure of these molecules. Theseperturbations can include insertions, deletions, substitutions and thelike. Such evaluations are useful because perturbations in thenucleotide and amino acid sequences are observed in a large number ofproteins associated with a growth dysregulated phenotype (see, e.g.,Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, amutation in the sequence of 193P1E1B may be indicative of the presenceor promotion of a tumor. Such assays therefore have diagnostic andpredictive value where a mutation in 193P1E1B indicates a potential lossof function or increase in tumor growth.

A wide variety of assays for observing perturbations in nucleotide andamino acid sequences are well known in the art. For example, the sizeand structure of nucleic acid or amino acid sequences of 193P1E1B geneproducts are observed by the Northern, Southern, Western, PCR and DNAsequencing protocols discussed herein. In addition, other methods forobserving perturbations in nucleotide and amino acid sequences such assingle strand conformation polymorphism analysis are well known in theart (see, e.g., U.S. Pat. Nos. 5,382,510 issued Sep. 7, 1999, and5,952,170 issued Jan. 17, 1995).

Additionally, one can examine the methylation status of a 193P1E1B genein a biological sample. Aberrant demethylation and/or hypermethylationof CpG islands in gene 5′ regulatory regions frequency occurs inimmortalized and transformed cells, and can result in altered expressionof various genes. For example, promoter hypermethylation of the pi-classglutathione S-transferase (a protein expressed in normal prostate butnot expressed in >90% of prostate carcinomas) appears to permanentlysilence transcription of this gene and is the most frequently detectedgenomic alteration in prostate carcinomas (De Marzo et al., Am. J.Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration ispresent in at least 70% of cases of high-grade prostatic intraepithelialneoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev.,1998, 7:531-536). In another example, expression of the LAGE-I tumorspecific gene (which is not expressed in normal prostate but isexpressed in 25-50% of prostate cancers) is induced by deoxy-azacytidinein lymphoblastoid cells, suggesting that tumoral expression is due todemethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). Avariety of assays for examining methylation status of a gene are wellknown in the art. For example, one can utilize, in Southernhybridization approaches, methylation-sensitive restriction enzymes thatcannot cleave sequences that contain methylated CpG sites to assess themethylation status of CpG islands. In addition, MSP (methylationspecific PCR) can rapidly profile the methylation status of all the CpGsites present in a CpG island of a given gene. This procedure involvesinitial modification of DNA by sodium bisulfite (which will convert allunmethylated cytosines to uracil) followed by amplification usingprimers specific for methylated versus unmethylated DNA. Protocolsinvolving methylation interference can also be found for example inCurrent Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel etat. eds., 1995.

Gene amplification is an additional method for assessing the status of193P1E1B. Gene amplification is measured in a sample directly, forexample, by conventional Southern blotting or Northern blotting toquantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad.Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies are employed thatrecognize specific duplexes, including DNA duplexes, RNA duplexes, andDNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turnare labeled and the assay carried out where the duplex is bound to asurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for thepresence of cancer cells using for example, Northern, dot blot or RT-PCRanalysis to detect 193P1E1B expression. The presence of RT-PCRamplifiable 193P1E1B mRNA provides an indication of the presence ofcancer. RT-PCR assays are well known in the art. RT-PCR detection assaysfor tumor cells in peripheral blood are currently being evaluated foruse in the diagnosis and management of a number of human solid tumors.In the prostate cancer field, these include RT-PCR assays for thedetection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol.Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect of the invention is an assessment of the susceptibilitythat an individual has for developing cancer. In one embodiment, amethod for predicting susceptibility to cancer comprises detecting193P1E1B mRNA or 193P1E1B protein in a tissue sample, its presenceindicating susceptibility to cancer, wherein the degree of 193P1E1B mRNAexpression correlates to the degree of susceptibility. In a specificembodiment, the presence of 193P1E1B in prostate or other tissue isexamined, with the presence of 193P1E1B in the sample providing anindication of prostate cancer susceptibility (or the emergence orexistence of a prostate tumor). Similarly, one can evaluate theintegrity 193P1E1B nucleotide and amino acid sequences in a biologicalsample, in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like. Thepresence of one or more perturbations in 193P1E1B gene products in thesample is an indication of cancer susceptibility (or the emergence orexistence of a tumor).

The invention also comprises methods for gauging tumor aggressiveness.In one embodiment, a method for gauging aggressiveness of a tumorcomprises determining the level of 193P1E1B mRNA or 193P1E1B proteinexpressed by tumor cells, comparing the level so determined to the levelof 193P1E1B mRNA or 193P1E1B protein expressed in a corresponding normaltissue taken from the same individual or a normal tissue referencesample, wherein the degree of 193P1E1B mRNA or 193P1E1B proteinexpression in the tumor sample relative to the normal sample indicatesthe degree of aggressiveness. In a specific embodiment, aggressivenessof a tumor is evaluated by determining the extent to which 193P1E1B isexpressed in the tumor cells, with higher expression levels indicatingmore aggressive tumors. Another embodiment is the evaluation of theintegrity of 193P1E1B nucleotide and amino acid sequences in abiological sample, in order to identify perturbations in the structureof these molecules such as insertions, deletions, substitutions and thelike. The presence of one or more perturbations indicates moreaggressive tumors.

Another embodiment of the invention is directed to methods for observingthe progression of a malignancy in an individual over time. In oneembodiment, methods for observing the progression of a malignancy in anindividual over time comprise determining the level of 193P1E1B mRNA or193P1E1B protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of 193P1E1B mRNA or 193P1E1Bprotein expressed in an equivalent tissue sample taken from the sameindividual at a different time, wherein the degree of 193P1E1B mRNA or193P1E1B protein expression in the tumor sample over time providesinformation on the progression of the cancer. In a specific embodiment,the progression of a cancer is evaluated by determining 193P1E1Bexpression in the tumor cells over time, where increased expression overtime indicates a progression of the cancer. Also, one can evaluate theintegrity 193P1E1B nucleotide and amino acid sequences in a biologicalsample in order to identify perturbations in the structure of thesemolecules such as insertions, deletions, substitutions and the like,where the presence of one or more perturbations indicates a progressionof the cancer.

The above diagnostic approaches can be combined with any one of a widevariety of prognostic and diagnostic protocols known in the art. Forexample, another embodiment of the invention is directed to methods forobserving a coincidence between the expression of 193P1E1B gene and193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1Bgene products) and a factor that is associated with malignancy, as ameans for diagnosing and prognosticating the status of a tissue sample.A wide variety of factors associated with malignancy can be utilized,such as the expression of genes associated with malignancy (e.g. PSA,PSCA and PSM expression for prostate cancer etc.) as well as grosscytological observations (see, e.g., Bocking et al., 1984, Anal. Quant.Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson etal., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg.Pathol. 23(8):918-24). Methods for observing a coincidence between theexpression of 193P1E1B gene and 193P1E1B gene products (or perturbationsin 193P1E1B gene and 193P1E1B gene products) and another factor that isassociated with malignancy are useful, for example, because the presenceof a set of specific factors that coincide with disease providesinformation crucial for diagnosing and prognosticating the status of atissue sample.

In one embodiment, methods for observing a coincidence between theexpression of 193P1E1B gene and 193P1E1B gene products (or perturbationsin 193P1E1B gene and 193P1E1B gene products) and another factorassociated with malignancy entails detecting the overexpression of193P1E1B mRNA or protein in a tissue sample, detecting theoverexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSMexpression), and observing a coincidence of 193P1E1B mRNA or protein andPSA mRNA or protein overexpression (or PSCA or PSM expression). In aspecific embodiment, the expression of 193P1E1B and PSA mRNA in prostatetissue is examined, where the coincidence of 193P1E1B and PSA mRNAoverexpression in the sample indicates the existence of prostate cancer,prostate cancer susceptibility or the emergence or status of a prostatetumor.

Methods for detecting and quantifying the expression of 193P1E1B mRNA orprotein are described herein, and standard nucleic acid and proteindetection and quantification technologies are well known in the art.Standard methods for the detection and quantification of 193P1E1B mRNAinclude in situ hybridization using labeled 193P1E1B riboprobes,Northern blot and related techniques using 193P1E1B polynucleotideprobes, RT-PCR analysis using primers specific for 193P1E1B, and otheramplification type detection methods, such as, for example, branchedDNA, SISBA, TMA and the like. In a specific embodiment,semi-quantitative RT-PCR is used to detect and quantify 193P1E1B mRNAexpression. Any number of primers capable of amplifying 193P1E1B can beused for this purpose, including but not limited to the various primersets specifically described herein. In a specific embodiment, polyclonalor monoclonal antibodies specifically reactive with the wild-type193P1E1B protein can be used in an immunohistochemical assay of biopsiedtissue.

IX.) Identification of Molecules That Interact With 193P1E1B

The 193P1E1B protein and nucleic acid sequences disclosed herein allow askilled artisan to identify proteins, small molecules and other agentsthat interact with 193P1E1B, as well as pathways activated by 193P1E1Bvia anyone of a variety of art accepted protocols. For example, one canutilize one of the so-called interaction trap systems (also referred toas the “two-hybrid assay”). In such systems, molecules interact andreconstitute a transcription factor which directs expression of areporter gene, whereupon the expression of the reporter gene is assayed.Other systems identify protein-protein interactions in vivo throughreconstitution of a eukaryotic transcriptional activator, see, e.g.,U.S. Pat. Nos. 5,955,280 issued Sep. 21, 1999, 5,925,523 issued Jul. 20,1999, 5,846,722 issued Dec. 8, 1998 and 6,004,746 issued Dec. 21, 1999.Algorithms are also available in the art for genome-based predictions ofprotein function (see, e.g., Marcotte, et al., Nature 402: Nov. 4, 1999,83-86).

Alternatively one can screen peptide libraries to identify moleculesthat interact with 193P1E1B protein sequences. In such methods, peptidesthat bind to 193P1E1B are identified by screening libraries that encodea random or controlled collection of amino acids. Peptides encoded bythe libraries are expressed as fusion proteins of bacteriophage coatproteins, the bacteriophage particles are then screened against the193P1E1B protein(s).

Accordingly, peptides having a wide variety of uses, such astherapeutic, prognostic or diagnostic reagents, are thus identifiedwithout any prior information on the structure of the expected ligand orreceptor molecule. Typical peptide libraries and screening methods thatcan be used to identify molecules that interact with 193P1E1B proteinsequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issuedMar. 3, 1998 and 5,733,731 issued Mar. 31, 1998.

Alternatively, cell lines that express 193P1E1B are used to identifyprotein-protein interactions mediated by 193P1E1B. Such interactions canbe examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 193P1E1Bprotein can be immunoprecipitated from 193P1E1B-expressing cell linesusing anti-193P1E1B antibodies. Alternatively, antibodies againstHis-tag can be used in a cell line engineered to express fusions of193P1E1B and a His-tag (vectors mentioned above). The immunoprecipitatedcomplex can be examined for protein association by procedures such asWestern blotting, ³⁵S-methionine labeling of proteins, proteinmicrosequencing, silver staining and two-dimensional gelelectrophoresis.

Small molecules and ligands that interact with 193P1E1B can beidentified through related embodiments of such screening assays. Forexample, small molecules can be identified that interfere with proteinfunction, including molecules that interfere with 193P1E1B's ability tomediate phosphorylation and de-phosphorylation, interaction with DNA orRNA molecules as an indication of regulation of cell cycles, secondmessenger signaling or tumorigenesis. Similarly, small molecules thatmodulate 193P1E1B-related ion channel, protein pump, or cellcommunication functions are identified and used to treat patients thathave a cancer that expresses 193P1E1B (see, e.g., Hille, B., IonicChannels of Excitable Membranes 2^(nd) Ed., Sinauer Assoc., Sunderland,Mass., 1992). Moreover, ligands that regulate 193P1E1B function can beidentified based on their ability to bind 193P1E1B and activate areporter construct. Typical methods are discussed for example in U.S.Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods for forminghybrid ligands in which at least one ligand is a small molecule. In anillustrative embodiment, cells engineered to express a fusion protein of193P1E1B and a DNA-binding protein are used to co-express a fusionprotein of a hybrid ligand/small molecule and a cDNA librarytranscriptional activator protein. The cells further contain a reportergene, the expression of which is conditioned on the proximity of thefirst and second fusion proteins to each other, an event that occursonly if the hybrid ligand binds to target sites on both hybrid proteins.Those cells that express the reporter gene are selected and the unknownsmall molecule or the unknown ligand is identified. This method providesa means of identifying modulators, which activate or inhibit 193P1E1B.

An embodiment of this invention comprises a method of screening for amolecule that interacts with a 193P1E1B amino acid sequence shown inFIG. 2 or FIG. 3, comprising the steps of contacting a population ofmolecules with a 193P1E1B amino acid sequence, allowing the populationof molecules and the 193P1E1B amino acid sequence to interact underconditions that facilitate an interaction, determining the presence of amolecule that interacts with the 193P1E1B amino acid sequence, and thenseparating molecules that do not interact with the 193P1E1B amino acidsequence from molecules that do. In a specific embodiment, the methodfurther comprises purifying, characterizing and identifying a moleculethat interacts with the 193P1E1B amino acid sequence. The identifiedmolecule can be used to modulate a function performed by 193P1E1B. In apreferred embodiment, the 193P1E1B amino acid sequence is contacted witha library of peptides.

X.) Therapeutic Methods and Compositions

The identification of 193P1E1B as a protein that is normally expressedin a restricted set of tissues, but which is also expressed in prostateand other cancers, opens a number of therapeutic approaches to thetreatment of such cancers. As contemplated herein, 193P1E1B functions asa transcription factor involved in activating tumor-promoting genes orrepressing genes that block tumorigenesis.

Accordingly, therapeutic approaches that inhibit the activity of a193P1E1B protein are useful for patients suffering from a cancer thatexpresses 193P1E1B. These therapeutic approaches generally fall into twoclasses. One class comprises various methods for inhibiting the bindingor association of a 193P1E1B protein with its binding partner or withother proteins. Another class comprises a variety of methods forinhibiting the transcription of a 193P1E1B gene or translation of193P1E1B mRNA.

X.A.) Anti-Cancer Vaccines

The invention provides cancer vaccines comprising a 193P1E1B-relatedprotein or 193P1E1B related nucleic acid. In view of the expression of193P1E1B, cancer vaccines prevent and/or treat 193P1E1B-expressingcancers with minimal or no effects on non-target tissues. The use of atumor antigen in a vaccine that generates humoral and/or cell-mediatedimmune responses as anti-cancer therapy is well known in the art and hasbeen employed in prostate cancer using human PSMA and rodent PAPimmunogens (Hodge et al., 1995, Int J. Cancer 63:231-237; Fong et al.,1997, J. Immunol. 159:3113-3117).

Such methods can be readily practiced by employing a 193P1E1B-relatedprotein, or a 193P1E1B-encoding nucleic acid molecule and recombinantvectors capable of expressing and presenting the 193P1E1B immunogen(which typically comprises a number of antibody or T cell epitopes).Skilled artisans understand that a wide variety of vaccine systems fordelivery of immunoreactive epitopes are known in the art (see, e.g.,Heryln et al., Ann Med 1999 Feb 31(1):66-78; Maruyama et al., CancerImmunol Immunother June 2000 49(3):123-32) Briefly, such methods ofgenerating an immune response (e.g. humoral and/or cell-mediated) in amammal, comprise the steps of: exposing the mammal's immune system to animmunoreactive epitope (e.g. an epitope present in a 193P1E1B proteinshown in FIG. 3 or analog or homolog thereof) so that the mammalgenerates an immune response that is specific for that epitope (e.g.generates antibodies that specifically recognize that epitope). In apreferred method, a 193P1E1B immunogen contains a biological motif, seee.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from193P1E1B indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.

The entire 193P1E1B protein, immunogenic regions or epitopes thereof canbe combined and delivered by various means. Such vaccine compositionscan include, for example, lipopeptides (e.g., Vitiello, A. et al., J.Clin. Invest. 95:341, 1995), peptide compositions encapsulated inpoly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge,et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptidecompositions contained in immune stimulating complexes (ISCOMS) (see,e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin ExpImmunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs)(see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988;Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated asmultivalent peptides; peptides for use in ballistic delivery systems,typically crystallized peptides, viral delivery vectors (Perkus, M. E.et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. etal., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda,P. K. et al., Virology 175:535, 1990), particles of viral or syntheticorigin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. etal., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R.,and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al.,Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol.148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked orparticle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993;Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993;Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S.H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev.Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16,1993). Toxin-targeted delivery technologies, also known as receptormediated targeting, such as those of Avant Immunotherapeutics, Inc.(Needham, Mass.) may also be used.

In patients with 193P1E1B-associated cancer, the vaccine compositions ofthe invention can also be used in conjunction with other treatments usedfor cancer, e.g., surgery, chemotherapy, drug therapies, radiationtherapies, etc. including use in combination with immune adjuvants suchas IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identifypeptides within 193P1E1B protein that bind corresponding HLA alleles(see e.g., Table IV; Epimer™ and Epimatrix™, Brown University; and,BIMAS, and SYFPEITHI. In a preferred embodiment, a 193P1E1B immunogencontains one or more amino acid sequences identified using techniqueswell known in the art, such as the sequences shown in Tables VIII-XXIand XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by anHLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or TableIV (E)) and/or a peptide of at least 9 amino acids that comprises an HLAClass II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As isappreciated in the art, the HLA Class I binding groove is essentiallyclosed ended so that peptides of only a particular size range can fitinto the groove and be bound, generally HLA Class I epitopes are 8, 9,10, or 11 amino acids long. In contrast, the HLA Class II binding grooveis essentially open ended; therefore a peptide of about 9 or more aminoacids can be bound by an HLA Class 11 molecule. Due to the bindinggroove differences between HLA Class I and 11, HLA Class I motifs arelength specific, i.e., position two of a Class I motif is the secondamino acid in an amino to carboxyl direction of the peptide. The aminoacid positions in a Class II motif are relative only to each other, notthe overall peptide, i.e., additional amino acids can be attached to theamino and/or carboxyl termini of a motif-bearing sequence. HLA Class IIepitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.

Antibody-Based Vaccines

A wide variety of methods for generating an immune response in a mammalare known in the art (for example as the first step in the generation ofhybridomas). Methods of generating an immune response in a mammalcomprise exposing the mammal's immune system to an immunogenic epitopeon a protein (e.g. a 193P1E1B protein) so that an immune response isgenerated. A typical embodiment consists of a method for generating animmune response to 193P1E1B in a host, by contacting the host with asufficient amount of at least one 193P1E1B B cell or cytotoxic T-cellepitope or analog thereof; and at least one periodic interval thereafterre-contacting the host with the 193P1E1B B cell or cytotoxic T-cellepitope or analog thereof. A specific embodiment consists of a method ofgenerating an immune response against a 193P1E1B-related protein or aman-made multiepitopic peptide comprising: administering 193P1E1Bimmunogen (e.g. a 193P1E1B protein or a peptide fragment thereof, a193P1E1B fusion protein or analog etc.) in a vaccine preparation to ahuman or another mammal. Typically, such vaccine preparations furthercontain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or auniversal helper epitope such as a PADRE™ peptide (Epimmune Inc., SanDiego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3);164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 andAlexander et al., Immunol. Res. 1998 18(2): 79-92). An alternativemethod comprises generating an immune response in an individual againsta 193P1E1B immunogen by: administering in vivo to muscle or skin of theindividual's body a DNA molecule that comprises a DNA sequence thatencodes a 193P1E1B immunogen, the DNA sequence operatively linked toregulatory sequences which control the expression of the DNA sequence;wherein the DNA molecule is taken up by cells, the DNA sequence isexpressed in the cells and an immune response is generated against theimmunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a geneticvaccine facilitator such as anionic lipids; saponins; lectins;estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; andurea is also administered. In addition, an antiidiotypic antibody can beadministered that mimics 193P1E1B, in order to generate a response tothe target antigen.

Nucleic Acid Vaccines:

Vaccine compositions of the invention include nucleic acid-mediatedmodalities. DNA or RNA that encode protein(s) of the invention can beadministered to a patient. Genetic immunization methods can be employedto generate prophylactic or therapeutic humoral and cellular immuneresponses directed against cancer cells expressing 193P1E1B. Constructscomprising DNA encoding a 193P1E1B-related protein/immunogen andappropriate regulatory sequences can be injected directly into muscle orskin of an individual, such that the cells of the muscle or skin take-upthe construct and express the encoded 193P1E1B protein/immunogen.Alternatively, a vaccine comprises a 193P1E1B-related protein.Expression of the 193P1E1B-related protein immunogen results in thegeneration of prophylactic or therapeutic humoral and cellular immunityagainst cells that bear a 193P1E1B protein. Various prophylactic andtherapeutic genetic immunization techniques known in the art can be used(for review, see information and references published at Internetaddress genweb.com). Nucleic acid-based delivery is described, forinstance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat.Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647;WO 98/04720. Examples of DNA-based delivery technologies include “nakedDNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery,cationic lipid complexes, and particle-mediated (“gene gun”) orpressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of theinvention can be expressed via viral or bacterial vectors. Various viralgene delivery systems that can be used in the practice of the inventioninclude, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol.8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)).Non-viral delivery systems can also be employed by introducing naked DNAencoding a 193P1E1B-related protein into the patent (e.g.,intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotidesequences that encode the peptides of the invention. Upon introductioninto a host, the recombinant vaccinia virus expresses the proteinimmunogenic peptide, and thereby elicits a host immune response.Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG(Bacille Calmelte Guerin). BCG vectors are described in Stover et al.,Nature 351:456-460 (1991). A wide variety of other vectors useful fortherapeutic administration or immunization of the peptides of theinvention, e.g. adeno and adeno-associated virus vectors, retroviralvectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, andthe like, will be apparent to those skilled in the art from thedescription herein.

Thus, gene delivery systems are used to deliver a 193P1E1B-relatednucleic add molecule. In one embodiment, the full-length human 193P1E1BcDNA is employed. In another embodiment, 193P1E1B nucleic acid moleculesencoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopesare employed.

Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immuneresponse. One approach involves the use of antigen presenting cells(APCS) such as dendritic cells (DC) to present 193P1E1B antigen to apatient's immune system. Dendritic cells express MHC class I and IImolecules, B7 co-stimulator, and IL-12, and are thus highly specializedantigen presenting cells. In prostate cancer, autologous dendritic cellspulsed with peptides of the prostate-specific membrane antigen (PSMA)are being used in a Phase I clinical trial to stimulate prostate cancerpatients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphyet al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used topresent 193P1E1B peptides to T cells in the context of MHC class I or IImolecules. In one embodiment, autologous dendritic cells are pulsed with193P1E1B peptides capable of binding to MHC class I and/or class IImolecules. In another embodiment, dendritic cells are pulsed with thecomplete 193P1E1B protein. Yet another embodiment involves engineeringthe overexpression of a 193P1E1B gene in dendritic cells using variousimplementing vectors known in the art, such as adenovirus (Arthur etal., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al.,1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNAtransfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), ortumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med.186:1177-1182). Cells that express 193P1E1B can also be engineered toexpress immune modulators, such as GM-CSF, and used as immunizingagents.

X.B.) 193P1E1B as a Target for Antibody-Based Therapy

193P1E1B is an attractive target for antibody-based therapeuticstrategies. A number of antibody strategies are known in the art fortargeting both extracellular and intracellular molecules (see, e.g.,complement and ADCC mediated killing as well as the use of intrabodies).Because 193P1E1B is expressed by cancer cells of various lineagesrelative to corresponding normal cells, systemic administration of193P1E1B-immunoreactive compositions are prepared that exhibit excellentsensitivity without toxic, non-specific and/or non-target effects causedby binding of the immunoreactive composition to non-target organs andtissues. Antibodies specifically reactive with domains of 193P1E1B areuseful to treat 193P1E1B-expressing cancers systemically, either asconjugates with a toxin or therapeutic agent, or as naked antibodiescapable of inhibiting cell proliferation or function.

193P1E1B antibodies can be introduced into a patient such that theantibody binds to 193P1E1B and modulates a function, such as aninteraction with a binding partner, and consequently mediatesdestruction of the tumor cells and/or inhibits the growth of the tumorcells. Mechanisms by which such antibodies exert a therapeutic effectcan include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulation of the physiological function of 193P1E1B,inhibition of ligand binding or signal transduction pathways, modulationof tumor cell differentiation, alteration of tumor angiogenesis factorprofiles, and/or apoptosis.

Those skilled in the art understand that antibodies can be used tospecifically target and bind immunogenic molecules such as animmunogenic region of a 193P1E1B sequence shown in FIG. 2 or FIG. 3. Inaddition, skilled artisans understand that it is routine to conjugateantibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:113678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents aredelivered directly to cells, such as by conjugating them to antibodiesspecific for a molecule expressed by that cell (e.g. 193P1E1B), thecytotoxic agent will exert its known biological effect (i.e.cytotoxicity) on those cells.

A wide variety of compositions and methods for using antibody-cytotoxicagent conjugates to kill cells are known in the art. In the context ofcancers, typical methods entail administering to an animal having atumor a biologically effective amount of a conjugate comprising aselected cytotoxic and/or therapeutic agent linked to a targeting agent(e.g. an anti-193P1E1B antibody) that binds to a marker (e.g. 193P1E1B)expressed, accessible to binding or localized on the cell surfaces. Atypical embodiment is a method of delivering a cytotoxic and/ortherapeutic agent to a cell expressing 193P1E1B, comprising conjugatingthe cytotoxic agent to an antibody that immunospecifically binds to a193P1E1B epitope, and, exposing the cell to the antibody-agentconjugate. Another illustrative embodiment is a method of treating anindividual suspected of suffering from metastasized cancer, comprising astep of administering parenterally to said individual a pharmaceuticalcomposition comprising a therapeutically effective amount of an antibodyconjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-193P1E1B antibodies can be done inaccordance with various approaches that have been successfully employedin the treatment of other types of cancer, including but not limited tocolon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138),multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari etal., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992,Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J.Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al.,1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994,Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin.Immunol. 11:117-127). Some therapeutic approaches involve conjugation ofnaked antibody to a toxin or radioisotope, such as the conjugation ofY⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDECPharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while othersinvolve co-administration of antibodies and other therapeutic agents,such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). Theantibodies can be conjugated to a therapeutic agent. To treat prostatecancer, for example, 193P1E1B antibodies can be administered inconjunction with radiation, chemotherapy or hormone ablation. Also,antibodies can be conjugated to a toxin such as calicheamicin (e.g.,Mylotarg™, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4kappa antibody conjugated to antitumor antibiotic calicheamicin) or amaytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform,ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).

Although 193P1E1B antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well. Fan et al. (Cancer Res.53:4637-4642, 1993), Prewett et al. (International J. of Onco.9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991)describe the use of various antibodies together with chemotherapeuticagents.

Although 193P1E1B antibody therapy is useful for all stages of cancer,antibody therapy can be particularly appropriate in advanced ormetastatic cancers. Treatment with the antibody therapy of the inventionis indicated for patients who have received one or more rounds ofchemotherapy. Alternatively, antibody therapy of the invention iscombined with a chemotherapeutic or radiation regimen for patients whohave not received chemotherapeutic treatment. Additionally, antibodytherapy can enable the use of reduced dosages of concomitantchemotherapy, particularly for patients who do not tolerate the toxicityof the chemotherapeutic agent very well.

Cancer patients can be evaluated for the presence and level of 193P1E1Bexpression, preferably using immunohistochemical assessments of tumortissue, quantitative 193P1E1B imaging, or other techniques that reliablyindicate the presence and degree of 193P1E1B expression.Immunohistochemical analysis of tumor biopsies or surgical specimens ispreferred for this purpose. Methods for immunohistochemical analysis oftumor tissues are well known in the art.

Anti-193P1E1B monoclonal antibodies that treat prostate and othercancers include those that initiate a potent immune response against thetumor or those that are directly cytotoxic. In this regard,anti-193P1E1B monoclonal antibodies (mAbs) can elicit tumor cell lysisby either complement-mediated or antibody-dependent cell cytotoxicity(ADCC) mechanisms, both of which require an intact Fc portion of theimmunoglobulin molecule for interaction with effector cell Fc receptorsites on complement proteins. In addition, anti-193P1E1B mAbs that exerta direct biological effect on tumor growth are useful to treat cancersthat express 193P1E1B. Mechanisms by which directly cytotoxic mAbs actinclude: inhibition of cell growth, modulation of cellulardifferentiation, modulation of tumor angiogenesis factor profiles, andthe induction of apoptosis. The mechanism(s) by which a particularanti-193P1E1B mAb exerts an anti-tumor effect is evaluated using anynumber of in vitro assays that evaluate cell death such as ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

In some patients, the use of murine or other non-human monoclonalantibodies, or human/mouse chimeric mAbs can induce moderate to strongimmune responses against the non-human antibody. This can result inclearance of the antibody from circulation and reduced efficacy. In themost severe cases, such an immune response can lead to the extensiveformation of immune complexes which, potentially, can cause renalfailure. Accordingly, preferred monoclonal antibodies used in thetherapeutic methods of the invention are those that are either fullyhuman or humanized and that bind specifically to the target 193P1E1Bantigen with high affinity but exhibit low or no antigenicity in thepatient.

Therapeutic methods of the invention contemplate the administration ofsingle anti-193P1E1B mAbs as well as combinations, or cocktails, ofdifferent mAbs. Such mAb cocktails can have certain advantages inasmuchas they contain mAbs that target different epitopes, exploit differenteffector mechanisms or combine directly cytotoxic mAbs with mAbs thatrely on immune effector functionality. Such mAbs in combination canexhibit synergistic therapeutic effects. In addition, anti-193P1E1B mAbscan be administered concomitantly with other therapeutic modalities,including but not limited to various chemotherapeutic agents,androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery orradiation. The anti-193P1E1B mAbs are administered in their “naked” orunconjugated form, or can have a therapeutic agent(s) conjugated tothem.

Anti-193P1E1B antibody formulations are administered via any routecapable of delivering the antibodies to a tumor cell. Routes ofadministration include, but are not limited to, intravenous,intraperitoneal, intramuscular, intratumor, intradermal, and the like.Treatment generally involves repeated administration of theanti-193P1E1B antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general,doses in the range of 10-1000 mg mAb per week are effective and welltolerated.

Based on clinical experience with the Herceptin™ mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kgIV of the anti-193P1E1B mAb preparation represents an acceptable dosingregimen. Preferably, the initial loading dose is administered as a90-minute or longer infusion. The periodic maintenance dose isadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. As appreciated by those of skill in the art,various factors can influence the ideal dose regimen in a particularcase. Such factors include, for example, the binding affinity and halflife of the Ab or mAbs used, the degree of 193P1E1B expression in thepatient, the extent of circulating shed 193P1E1B antigen, the desiredsteady-state antibody concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient.

Optionally, patients should be evaluated for the levels of 193P1E1B in agiven sample (e.g. the levels of circulating 193P1E1B antigen and/or193P1E1B expressing cells) in order to assist in the determination ofthe most effective dosing regimen, etc. Such evaluations are also usedfor monitoring purposes throughout therapy, and are useful to gaugetherapeutic success in combination with the evaluation of otherparameters (for example, urine cytology and/or ImmunoCyt levels inbladder cancer therapy, or by analogy, serum PSA levels in prostatecancer therapy).

Anti-idiotypic anti-193P1E1B antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga 193P1E1B-related protein. In particular, the generation ofanti-idiotypic antibodies is well known in the art; this methodology canreadily be adapted to generate anti-idiotypic anti-193P1E1B antibodiesthat mimic an epitope on a 193P1E1B-related protein (see, for example,Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin.Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother.43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccinestrategies.

X.C.) 193P1E1B as a Target for Cellular Immune Responses

Vaccines and methods of preparing vaccines that contain animmunogenically effective amount of one or more HLA-binding peptides asdescribed herein are further embodiments of the invention. Furthermore,vaccines in accordance with the invention encompass compositions of oneor more of the claimed peptides. A peptide can be present in a vaccineindividually. Alternatively, the peptide can exist as a homopolymercomprising multiple copies of the same peptide, or as a heteropolymer ofvarious peptides. Polymers have the advantage of increased immunologicalreaction and, where different peptide epitopes are used to make up thepolymer, the additional ability to induce antibodies and/or CTLs thatreact with different antigenic determinants of the pathogenic organismor tumor-related peptide targeted for an immune response. Thecomposition can be a naturally occurring region of an antigen or can beprepared, e.g., recombinantly or by chemical synthesis.

Carriers that can be used with vaccines of the invention are well knownin the art, and include, e.g., thyroglobulin, albumins such as humanserum albumin, tetanus toxoid, polyamino acids such as poly L-lysine,poly L-glutamic acid, influenza, hepatitis B virus core protein, and thelike. The vaccines can contain a physiologically tolerable (i.e.,acceptable) diluent such as water, or saline, preferably phosphatebuffered saline. The vaccines also typically include an adjuvant.Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate,aluminum hydroxide, or alum are examples of materials well known in theart. Additionally, as disclosed herein, CTL responses can be primed byconjugating peptides of the invention to lipids, such astripalmitoyl-S-glycerylcysteinlyseryl-serine (P₃CSS). Moreover, anadjuvant such as a syntheticcytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotideshas been found to increase CTL responses 10- to 100-fold. (see, e.g.Davila and Celis, J. Immunol. 165:539-547 (2000))

Upon immunization with a peptide composition in accordance with theinvention, via injection, aerosol, oral, transdermal, transmucosal,intrapleural, intrathecal, or other suitable routes, the immune systemof the host responds to the vaccine by producing large amounts of CTLsand/or HTLs specific for the desired antigen. Consequently, the hostbecomes at least partially immune to later development of cells thatexpress or overexpress 193P1E1B antigen, or derives at least sometherapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptidecomponents with components that induce or facilitate neutralizingantibody and or helper T cell responses directed to the target antigen.A preferred embodiment of such a composition comprises class I and classII epitopes in accordance with the invention. An alternative embodimentof such a composition comprises a class I and/or class II epitope inaccordance with the invention, along with a cross reactive HTL epitopesuch as PADRE™ (Epimmune, San Diego, Calif.) molecule (described e.g.,in U.S. Pat. No. 5,736,142).

A vaccine of the invention can also include antigen-presenting cells(APC), such as dendritic cells (DC), as a vehicle to present peptides ofthe invention. Vaccine compositions can be created in vitro, followingdendritic cell mobilization and harvesting, whereby loading of dendriticcells occurs in vitro. For example, dendritic cells are transfected,e.g., with a minigene in accordance with the invention, or are pulsedwith peptides. The dendritic cell can then be administered to a patientto elicit immune responses in vivo. Vaccine compositions, either DNA- orpeptide-based, can also be administered in vivo in combination withdendritic cell mobilization whereby loading of dendritic cells occurs invivo.

Preferably, the following principles are utilized when selecting anarray of epitopes for inclusion in a polyepitopic composition for use ina vaccine, or for selecting discrete epitopes to be included in avaccine and/or to be encoded by nucleic acids such as a minigene. It ispreferred that each of the following principles be balanced in order tomake the selection. The multiple epitopes to be incorporated in a givenvaccine composition may be, but need not be, contiguous in sequence inthe native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immuneresponses that have been observed to be correlated with tumor clearance.For HLA Class I this includes 3-4 epitopes that come from at least onetumor associated antigen (TM). For HLA Class II a similar rationale isemployed; again 34 epitopes are selected from at least one TAA (see,e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAAmay be used in combination with epitopes from one or more additionalTAAs to produce a vaccine that targets tumors with varying expressionpatterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinityestablished to be correlated with immunogenicity: for HLA Class I anIC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀of 1000 nM or less.

3.) Sufficient supermotif bearing-peptides, or a sufficient array ofallele-specific motif-bearing peptides, are selected to give broadpopulation coverage. For example, it is preferable to have at least 80%population coverage. A Monte Carlo analysis, a statistical evaluationknown in the art, can be employed to assess the breadth, or redundancyof, population coverage.

4.) When selecting epitopes from cancer-related antigens it is oftenuseful to select analogs because the patient may have developedtolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as “nestedepitopes.” Nested epitopes occur where at least two epitopes overlap ina given peptide sequence. A nested peptide sequence can comprise B cell,HLA class I and/or HLA class II epitopes. When providing nestedepitopes, a general objective is to provide the greatest number ofepitopes per sequence. Thus, an aspect is to avoid providing a peptidethat is any longer than the amino terminus of the amino terminal epitopeand the carboxyl terminus of the carboxyl terminal epitope in thepeptide. When providing a multi-epitopic sequence, such as a sequencecomprising nested epitopes, it is generally important to screen thesequence in order to insure that it does not have pathological or otherdeleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene,an objective is to generate the smallest peptide that encompasses theepitopes of interest. This principle is similar, if not the same as thatemployed when selecting a peptide comprising nested epitopes. However,with an artificial polyepitopic peptide, the size minimization objectiveis balanced against the need to integrate any spacer sequences betweenepitopes in the polyepitopic protein. Spacer amino acid residues can,for example, be introduced to avoid junctional epitopes (an epitoperecognized by the immune system, not present in the target antigen, andonly created by the man-made juxtaposition of epitopes), or tofacilitate cleavage between epitopes and thereby enhance epitopepresentation. Junctional epitopes are generally to be avoided becausethe recipient may generate an immune response to that non-nativeepitope. Of particular concern is a junctional epitope that is a“dominant epitope.” A dominant epitope may lead to such a zealousresponse that immune responses to other epitopes are diminished orsuppressed.

7.) Where the sequences of multiple variants of the same target proteinare present, potential peptide epitopes can also be selected on thebasis of their conservancy. For example, a criterion for conservancy maydefine that the entire sequence of an HLA class I binding peptide or theentire 9-mer core of a class II binding peptide be conserved in adesignated percentage of the sequences evaluated for a specific proteinantigen.

X.C.1. Minigene Vaccines

A number of different approaches are available which allow simultaneousdelivery of multiple epitopes. Nucleic acids encoding the peptides ofthe invention are a particularly useful embodiment of the invention.Epitopes for inclusion in a minigene are preferably selected accordingto the guidelines set forth in the previous section. A preferred meansof administering nucleic acids encoding the peptides of the inventionuses minigene constructs encoding a peptide comprising one or multipleepitopes of the invention.

The use of multi-epitope minigenes is described below and in, Ishioka etal., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J. L., J.Virol. 71:2292, 1997; Thomson, S. A. et al., J. Immunol. 157:822, 1996;Whitton, J. L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine16:426, 1998. For example, a multi-epitope DNA plasmid encodingsupermotif-and/or motif-bearing epitopes derived 193P1E1B, the PADRE®universal helper T cell epitope or multiple HTL epitopes from 193P1E1B(see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmicreticulum-translocating signal sequence can be engineered. A vaccine mayalso comprise epitopes that are derived from other TAAs.

The immunogenicity of a multi-epitopic minigene can be confirmed intransgenic mice to evaluate the magnitude of CTL induction responsesagainst the epitopes tested. Further, the immunogenicity of DNA-encodedepitopes in vivo can be correlated with the in vitro responses ofspecific CTL lines against target cells transfected with the DNAplasmid. Thus, these experiments can show that the minigene serves toboth: 1.) generate a CTL response and 2.) that the induced CTLsrecognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes(minigene) for expression in human cells, the amino acid sequences ofthe epitopes may be reverse translated. A human codon usage table can beused to guide the codon choice for each amino acid. Theseepitope-encoding DNA sequences may be directly adjoined, so that whentranslated, a continuous polypeptide sequence is created. To optimizeexpression and/or immunogenicity, additional elements can beincorporated into the minigene design. Examples of amino acid sequencesthat can be reverse translated and included in the minigene sequenceinclude: HLA class I epitopes, HLA class II epitopes, antibody epitopes,a ubiquitination signal sequence, and/or an endoplasmic reticulumtargeting signal. In addition, HLA presentation of CTL and HTL epitopesmay be improved by including synthetic (e.g. poly-alanine) ornaturally-occurring flanking sequences adjacent to the CTL or HTLepitopes; these larger peptides comprising the epitope(s) are within thescope of the invention.

The minigene sequence may be converted to DNA by assemblingoligonucleotides that encode the plus and minus strands of the minigene.Overlapping oligonucleotides (30-100 bases long) may be synthesized,phosphorylated, purified and annealed under appropriate conditions usingwell known techniques. The ends of the oligonucleotides can be joined,for example, using T4 DNA ligase. This synthetic minigene, encoding theepitope polypeptide, can then be cloned into a desired expressionvector.

Standard regulatory sequences well known to those of skill in the artare preferably included in the vector to ensure expression in the targetcells. Several vector elements are desirable: a promoter with adown-stream cloning site for minigene insertion; a polyadenylationsignal for efficient transcription termination; an E. coli origin ofreplication; and an E. coli selectable marker (e.g. ampicillin orkanamycin resistance). Numerous promoters can be used for this purpose,e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat.Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Additional vector modifications may be desired to optimize minigeneexpression and immunogenicity. In some cases, introns are required forefficient gene expression, and one or more synthetic ornaturally-occurring introns could be incorporated into the transcribedregion of the minigene. The inclusion of mRNA stabilization sequencesand sequences for replication in mammalian cells may also be consideredfor increasing minigene expression.

Once an expression vector is selected, the minigene is cloned into thepolylinker region downstream of the promoter. This plasmid istransformed into an appropriate E. coli strain, and DNA is preparedusing standard techniques. The orientation and DNA sequence of theminigene, as well as all other elements included in the vector, areconfirmed using restriction mapping and DNA sequence analysis. Bacterialcells harboring the correct plasmid can be stored as a master cell bankand a working cell bank.

In addition, immunostimulatory sequences (ISSs or CpGs) appear to play arole in the immunogenicity of DNA vaccines. These sequences may beincluded in the vector, outside the minigene coding sequence, if desiredto enhance immunogenicity.

In some embodiments, a bi-cistronic expression vector which allowsproduction of both the minigene-encoded epitopes and a second protein(included to enhance or decrease immunogenicity) can be used. Examplesof proteins or polypeptides that could beneficially enhance the immuneresponse if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF),cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, orfor HTL responses, pan-DR binding proteins (PADRE™, Epimmune, San Diego,Calif.). Helper (HTL) epitopes can be joined to intracellular targetingsignals and expressed separately from expressed CTL epitopes; thisallows direction of the HTL epitopes to a cell compartment differentthan that of the CTL epitopes. If required, this could facilitate moreefficient entry of HTL epitopes into the HLA class II pathway, therebyimproving HTL induction. In contrast to HTL or CTL induction,specifically decreasing the immune response by co-expression ofimmunosuppressive molecules (e.g. TGF-β) may be beneficial in certaindiseases.

Therapeutic quantities of plasmid DNA can be produced for example, byfermentation in E. coli, followed by purification. Aliquots from theworking cell bank are used to inoculate growth medium, and grown tosaturation in shaker flasks or a bioreactor according to well-knowntechniques. Plasmid DNA can be purified using standard bioseparationtechnologies such as solid phase anion-exchange resins supplied byQIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can beisolated from the open circular and linear forms using gelelectrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety offormulations. The simplest of these is reconstitution of lyophilized DNAin sterile phosphate-buffer saline (PBS). This approach, known as “nakedDNA,” is currently being used for intramuscular (IM) administration inclinical trials. To maximize the immunotherapeutic effects of minigeneDNA vaccines, an alternative method for formulating purified plasmid DNAmay be desirable. A variety of methods have been described, and newtechniques may become available. Cationic lipids, glycolipids, andfusogenic liposomes can also be used in the formulation (see, e.g., asdescribed by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Pat No. 5,279,833; WO 91/06309; and Feigner, et al.,Proc. Nat'l. Acad. Sci. USA 84:7413 (1987). In addition, peptides andcompounds referred to collectively as protective, interactive,non-condensing compounds (PINC) could also be complexed to purifiedplasmid DNA to influence variables such as stability, intramusculardispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay forexpression and HLA class I presentation of minigene-encoded CTLepitopes. For example, the plasmid DNA is introduced into a mammaliancell line that is suitable as a target for standard CTL chromium releaseassays. The transfection method used will be dependent on the finalformulation. Electroporation can be used for “naked” DNA, whereascationic lipids allow direct in vitro transfection. A plasmid expressinggreen fluorescent protein (GFP) can be co-transfected to allowenrichment of transfected cells using fluorescence activated cellsorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and usedas target cells for epitope-specific CTL lines; cytolysis, detected by⁵¹Cr release, indicates both production of, and HLA presentation of,minigene-encoded CTL epitopes. Expression of HTL epitopes may beevaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing ofminigene DNA formulations. Transgenic mice expressing appropriate humanHLA proteins are immunized with the DNA product. The dose and route ofadministration are formulation dependent (e.g., IM for DNA in PBS,intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days afterimmunization, splenocytes are harvested and restimulated for one week inthe presence of peptides encoding each epitope being tested. Thereafter,for CTL effector cells, assays are conducted for cytolysis ofpeptide-loaded, ⁵¹Cr-labeled target cells using standard techniques.Lysis of target cells that were sensitized by HLA loaded with peptideepitopes, corresponding to minigene-encoded epitopes, demonstrates DNAvaccine function for in vivo induction of CTLs. Immunogenicity of HTLepitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballisticdelivery as described, for instance, in U.S. Pat. No. 5,204,253. Usingthis technique, particles comprised solely of DNA are administered. In afurther alternative embodiment, DNA can be adhered to particles, such asgold particles.

Minigenes can also be delivered using other bacterial or viral deliverysystems well known in the art, e.g., an expression construct encodingepitopes of the invention can be incorporated into a viral vector suchas vaccinia.

XC.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising CTL peptides of the invention can bemodified, e.g., analoged, to provide desired attributes, such asimproved serum half life, broadened population coverage or enhancedimmunogenicity.

For instance, the ability of a peptide to induce CTL activity can beenhanced by linking the peptide to a sequence which contains at leastone epitope that is capable of inducing a T helper cell response.Although a CTL peptide can be directly linked to a T helper peptide,often CTL epitope/HTL epitope conjugates are linked by a spacermolecule. The spacer is typically comprised of relatively small, neutralmolecules, such as amino acids or amino acid mimetics, which aresubstantially uncharged under physiological conditions. The spacers aretypically selected from, e.g., Ala, Gly, or other neutral spacers ofnonpolar amino acids or neutral polar amino acids. It will be understoodthat the optionally present spacer need not be comprised of the sameresidues and thus may be a hetero- or homo-oligomer. When present, thespacer will usually be at least one or two residues, more usually threeto six residues and sometimes 10 or more residues. The CTL peptideepitope can be linked to the T helper peptide epitope either directly orvia a spacer either at the amino or carboxy terminus of the CTL peptide.The amino terminus of either the immunogenic peptide or the T helperpeptide may be acylated.

In certain embodiments, the T helper peptide is one that is recognizedby T helper cells present in a majority of a genetically diversepopulation. This can be accomplished by selecting peptides that bind tomany, most, or all of the HLA class II molecules. Examples of such aminoacid bind many HLA Class II molecules include sequences from antigenssuch as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO:48), Plasmodium falciparum circumsporozoite (CS) protein at positions378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 49), and Streptococcus 18 kDprotein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 50). Otherexamples include peptides bearing a DR 14-7 supermotif, or either of theDR3 motifs.

Alternatively, it is possible to prepare synthetic peptides capable ofstimulating T helper lymphocytes, in a loosely HLA-restricted fashion,using amino acid sequences not found in nature (see, e.g., PCTpublication WO 95/07707). These synthetic compounds calledPan-DR-binding epitopes (e.g., PADRE™, Epimmune, Inc., San Diego,Calif.) are designed, most preferably, to bind most HLA-DR (human HLAclass II) molecules. For instance, a pan-DR-binding epitope peptidehaving the formula: AKXVAAWTLKAAA (SEQ ID NO: 51), where a is eithercyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanineor L-alanine, has been found to bind to most HLA-DR alleles, and tostimulate the response of T helper lymphocytes from most individuals,regardless of their HLA type. An alternative of a pan-DR binding epitopecomprises all “L” natural amino acids and can be provided in the form ofnucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biologicalproperties. For example, they can be modified to include D-amino acidsto increase their resistance to proteases and thus extend their serumhalf life, or they can be conjugated to other molecules such as lipids,proteins, carbohydrates, and the like to increase their biologicalactivity. For example, a T helper peptide can be conjugated to one ormore palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents

In some embodiments it may be desirable to include in the pharmaceuticalcompositions of the invention at least one component which primes Blymphocytes or T lymphocytes. Lipids have been identified as agentscapable of priming CTL in vivo. For example, palmitic acid residues canbe attached to the ε- and α-amino groups of a lysine residue and thenlinked, e.g., via one or more linking residues such as Gly, Gly-Gly-,Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidatedpeptide can then be administered either directly in a micelle orparticle, incorporated into a liposome, or emulsified in an adjuvant,e.g., incomplete Freund's adjuvant. In a preferred embodiment, aparticularly effective immunogenic composition comprises palmitic acidattached to ε- and α-amino groups of Lys, which is attached via linkage,e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. colilipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine(P₃CSS) can be used to prime virus specific CTL when covalently attachedto an appropriate peptide (see, e.g., Deres, et al., Nature 342:561,1989). Peptides of the invention can be coupled to P₃CSS, for example,and the lipopeptide administered to an individual to prime specificallyan immune response to the target antigen. Moreover, because theinduction of neutralizing antibodies can also be primed withP3CSS-conjugated epitopes, two such compositions can be combined to moreeffectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTLPeptides

An embodiment of a vaccine composition in accordance with the inventioncomprises ex vivo administration of a cocktail of epitope-bearingpeptides to PBMC, or isolated DC therefrom, from the patient's blood. Apharmaceutical to facilitate harvesting of DC can be used, such asProgenipoietin™ (Pharmacia-Monsanto, St. Louis, Mo.) or GM-CSF/IL-4.After pulsing the DC with peptides and prior to reinfusion intopatients, the DC are washed to remove unbound peptides. In thisembodiment, a vaccine comprises peptide-pulsed DCs which present thepulsed peptide epitopes complexed with HLA molecules on their surfaces.

The DC can be pulsed ex vivo with a cocktail of peptides, some of whichstimulate CTL responses to 193P1E1B. Optionally, a helper T cell (HTL)peptide, such as a natural or artificial loosely restricted HLA Class IIpeptide, can be included to facilitate the CTL response. Thus, a vaccinein accordance with the invention is used to treat a cancer whichexpresses or overexpresses 193P1E1B.

X.D. Adoptive Immunotherapy

Antigenic 193P1E1B-related peptides are used to elicit a CTL and/or HTLresponse ex vivo, as well. The resulting CTL or HTL cells, can be usedto treat tumors in patients that do not respond to other conventionalforms of therapy, or will not respond to a therapeutic vaccine peptideor nucleic acid in accordance with the invention. Ex vivo CTL or HTLresponses to a particular antigen are induced by incubating in tissueculture the patient's, or genetically compatible, CTL or HTL precursorcells together with a source of antigen-presenting cells (APC), such asdendritic cells, and the appropriate immunogenic peptide. After anappropriate incubation time (typically about 7-28 days), in which theprecursor cells are activated and expanded into effector cells, thecells are infused back into the patient, where they will destroy (CTL)or facilitate destruction (HTL) of their specific target cell (e.g., atumor cell). Transfected dendritic cells may also be used as antigenpresenting cells.

X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions of the invention are typicallyused to treat and/or prevent a cancer that expresses or overexpresses193P1E1B. In therapeutic applications, peptide and/or nucleic acidcompositions are administered to a patient in an amount sufficient toelicit an effective B cell, CTL and/or HTL response to the antigen andto cure or at least partially arrest or slow symptoms and/orcomplications. An amount adequate to accomplish this is defined as“therapeutically effective dose.” Amounts effective for this use willdepend on, e.g., the particular composition administered, the manner ofadministration, the stage and severity of the disease being treated, theweight and general state of health of the patient, and the judgment ofthe prescribing physician.

For pharmaceutical compositions, the immunogenic peptides of theinvention, or DNA encoding them, are generally administered to anindividual already bearing a tumor that expresses 193P1E1B. The peptidesor DNA encoding them can be administered individually or as fusions ofone or more peptide sequences. Patients can be treated with theimmunogenic peptides separately or in conjunction with other treatments,such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the firstdiagnosis of 193P1E1B-associated cancer. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. The embodiment of the vaccine composition (i.e., including,but not limited to embodiments such as peptide cocktails, polyepitopicpolypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells)delivered to the patient may vary according to the stage of the diseaseor the patient's health status. For example, in a patient with a tumorthat expresses 193P1E1B, a vaccine comprising 193P1E1B-specific CTL maybe more efficacious in killing tumor cells in patient with advanceddisease than alternative embodiments.

It is generally important to provide an amount of the peptide epitopedelivered by a mode of administration sufficient to stimulateeffectively a cytotoxic T cell response; compositions which stimulatehelper T cell responses can also be given in accordance with thisembodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in aunit dosage range where the lower value is about 1, 5, 50, 500, or 1,000μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg.Dosage values for a human typically range from about 500 μg to about50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0μg to about 50,000 μg of peptide pursuant to a boosting regimen overweeks to months may be administered depending upon the patient'sresponse and condition as determined by measuring the specific activityof CTL and HTL obtained from the patient's blood. Administration shouldcontinue until at least clinical symptoms or laboratory tests indicatethat the neoplasia, has been eliminated or reduced and for a periodthereafter. The dosages, routes of administration, and dose schedulesare adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the presentinvention are employed in serious disease states, that is,life-threatening or potentially life threatening situations. In suchcases, as a result of the minimal amounts of extraneous substances andthe relative nontoxic nature of the peptides in preferred compositionsof the invention, it is possible and may be felt desirable by thetreating physician to administer substantial excesses of these peptidecompositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely asprophylactic agents. Generally the dosage for an initial prophylacticimmunization generally occurs in a unit dosage range where the lowervalue is about 1, 5, 50, 500, or 1000 μg and the higher value is about10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a humantypically range from about 500 μg to about 50,000 μg per 70 kilogrampatient. This is followed by boosting dosages of between about 1.0 μg toabout 50,000 μg of peptide administered at defined intervals from aboutfour weeks to six months after the initial administration of vaccine.The immunogenicity of the vaccine can be assessed by measuring thespecific activity of CTL and HTL obtained from a sample of the patient'sblood.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral, nasal, intrathecal, or local (e.g. as acream or topical ointment) administration. Preferably, thepharmaceutical compositions are administered parentally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the immunogenic peptides dissolved or suspendedin an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e.g., water, buffered water,0.8% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well-known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH-adjusting and buffering agents, tonicity adjusting agents, wettingagents, preservatives, and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sorbitanmonolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceuticalformulations can vary widely, i.e., from less than about 0.1%, usuallyat or at least about 2% to as much as 20% to 50% or more by weight, andwill be selected primarily by fluid volumes, viscosities, etc., inaccordance with the particular mode of administration selected.

A human unit dose form of a composition is typically included in apharmaceutical composition that comprises a human unit dose of anacceptable carrier, in one embodiment an aqueous carrier, and isadministered in a volume/quantity that is known by those of skill in theart to be used for administration of such compositions to humans (see,e.g., Remington's Pharmaceutical Sciences, 17^(th) Edition, A. Gennaro,Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptidedose for initial immunization can be from about 1 to about 50,000 μ,generally 100-5,000 μg, for a 70 kg patient. For example, for nucleicacids an initial immunization may be performed using an expressionvector in the form of naked nucleic acid administered IM (or SC or ID)in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to1000 μg) can also be administered using a gene gun. Following anincubation period of 3-4 weeks, a booster dose is then administered. Thebooster can be recombinant fowlpox virus administered at a dose of 5-10⁷to 5×10⁹ pfu.

For antibodies, a treatment generally involves repeated administrationof the anti-193P1E1B antibody preparation, via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. In general,doses in the range of 10-500 mg mAb per week are effective and welltolerated. Moreover, an initial loading dose of approximately 4 mg/kgpatient body weight IV, followed by weekly doses of about 2 mg/kg IV ofthe anti-193P1E1B mAb preparation represents an acceptable dosingregimen. As appreciated by those of skill in the art, various factorscan influence the ideal dose in a particular case. Such factors include,for example, half life of a composition, the binding affinity of an Ab,the immunogenicity of a substance, the degree of 193P1E1B expression inthe patient, the extent of circulating shed 193P1E1B antigen, thedesired steady-state concentration level, frequency of treatment, andthe influence of chemotherapeutic or other agents used in combinationwith the treatment method of the invention, as well as the health statusof a particular patient. Non-limiting preferred human unit doses are,for example, 500 μg -1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200mg-300 mg, 400 mg-500 mg, 500 mg-600 mg, 600 mg -700 mg, 700 mg-800 mg,800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, thedose is in a range of 2-5 mg/kg body weight, e.g., with follow on weeklydoses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg bodyweight followed, e.g., in two, three or four weeks by weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two, three or four weeks byweekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m² of body areaweekly; 1-600 mg m² of body area weekly; 225-400 mg m² of body areaweekly; these does can be followed by weekly doses for 2, 3, 4, 5, 6, 7,8, 9, 19, 11, 12 or more weeks.

In one embodiment, human unit dose forms of polynucleotides comprise asuitable dosage range or effective amount that provides any therapeuticeffect. As appreciated by one of ordinary skill in the art a therapeuticeffect depends on a number of factors, including the sequence of thepolynucleotide, molecular weight of the polynucleotide and route ofadministration. Dosages are generally selected by the physician or otherhealth care professional in accordance with a variety of parametersknown in the art, such as severity of symptoms, history of the patientand the like. Generally, for a polynucleotide of about 20 bases, adosage range may be selected from, for example, an independentlyselected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to anindependently selected upper limit, greater than the lower limit, ofabout 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dosemay be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg,0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg, or 500 to 10,000 mg/kg.Generally, parenteral routes of administration may require higher dosesof polynucleotide compared to more direct application to the nucleotideto diseased tissue, as do polynucleotides of increasing length.

In one embodiment, human unit dose forms of T-cells comprise a suitabledosage range or effective amount that provides any therapeutic effect.As appreciated by one of ordinary skill in the art, a therapeutic effectdepends on a number of factors. Dosages are generally selected by thephysician or other health care professional in accordance with a varietyof parameters known in the art, such as severity of symptoms, history ofthe patient and the like. A dose may be about 10⁴ cells to about 10⁶cells, about 10⁶ cells to about 10⁸ cells, about 10⁸ to about 10¹¹cells, or about 10⁸ to about 5×10¹⁰ cells. A dose may also about 10⁶cells/m² to about 10¹⁰ cells/m², or about 10⁶ cells/m² to about 10⁸cells/m².

Proteins(s) of the invention, and/or nucleic acids encoding theprotein(s), can also be administered via liposomes, which may also serveto: 1) target the proteins(s) to a particular tissue, such as lymphoidtissue; 2) to target selectively to diseases cells; or, 3) to increasethe half-life of the peptide composition. Liposomes include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, thepeptide to be delivered is incorporated as part of a liposome, alone orin conjunction with a molecule which binds to a receptor prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes either filled or decorated with a desired peptide of theinvention can be directed to the site of lymphoid cells, where theliposomes then deliver the peptide compositions. Liposomes for use inaccordance with the invention are formed from standard vesicle-forminglipids, which generally include neutral and negatively chargedphospholipids and a sterol, such as cholesterol. The selection of lipidsis generally guided by consideration of, e.g., liposome size, acidlability and stability of the liposomes in the blood stream. A varietyof methods are available for preparing liposomes, as described in, e.g.,Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat.Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporatedinto the liposome can include, e.g., antibodies or fragments thereofspecific for cell surface determinants of the desired immune systemcells. A liposome suspension containing a peptide may be administeredintravenously, locally, topically, etc. in a dose which varies accordingto, inter alia, the manner of administration, the peptide beingdelivered, and the stage of the disease being treated.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, immunogenic peptides are preferably suppliedin finely divided form along with a surfactant and propellant. Typicalpercentages of peptides are about 0.01 %-20% by weight, preferably about1%-10%. The surfactant must, of course, be nontoxic, and preferablysoluble in the propellant. Representative of such agents are the estersor partial esters of fatty acids containing from about 6 to 22 carbonatoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic,linolenic, olesteric and oleic acids with an aliphatic polyhydricalcohol or its cyclic anhydride. Mixed esters, such as mixed or naturalglycerides may be employed. The surfactant may constitute about 0.1%-20%by weight of the composition, preferably about 0.25-5%. The balance ofthe composition is ordinarily propellant. A carrier can also beincluded, as desired, as with, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of 193P1E1B.

As disclosed herein, 193P1E1B polynucleotides, polypeptides, reactivecytotoxic T cells (CTL), reactive helper T cells (HTL) andanti-polypeptide antibodies are used in well known diagnostic,prognostic and therapeutic assays that examine conditions associatedwith dysregulated cell growth such as cancer, in particular the cancerslisted in Table I (see, e.g., both its specific pattern of tissueexpression as well as its overexpression in certain cancers as describedfor example in the Example entitled “Expression analysis of 193P1E1B innormal tissues, and patient specimens”).

193P1E1B can be analogized to a prostate associated antigen PSA, thearchetypal marker that has been used by medical practitioners for yearsto identify and monitor the presence of prostate cancer (see, e.g.,Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J.Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. CancerInst. 91(19): 1635-1640(1999)). A variety of other diagnostic markersare also used in similar contexts including p53 and K-ras (see, e.g.,Tulchinsky et al., Int J Mol Med Jul. 4, 1999 (1):99-102 and Minimoto etal., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of193P1E1B polynucleotides and polypeptides (as well as 193P1E1Bpolynucleotide probes and anti-193P1E1B antibodies used to identify thepresence of these molecules) and their properties allows skilledartisans to utilize these molecules in methods that are analogous tothose used, for example, in a variety of diagnostic assays directed toexamining conditions associated with cancer.

Typical embodiments of diagnostic methods which utilize the 193P1E1Bpolynucleotides, polypeptides, reactive T cells and antibodies areanalogous to those methods from well-established diagnostic assays,which employ, e.g., PSA polynucleotides, polypeptides, reactive T cellsand antibodies. For example, just as PSA polynucleotides are used asprobes (for example in Northern analysis, see, e.g., Sharief et al.,Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example inPCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190(2000)) to observe the presence and/or the level of PSA mRNAs in methodsof monitoring PSA overexpression or the metastasis of prostate cancers,the 193P1E1B polynucleotides described herein can be utilized in thesame way to detect 193P1E1B overexpression or the metastasis of prostateand other cancers expressing this gene. Alternatively, just as PSApolypeptides are used to generate antibodies specific for PSA which canthen be used to observe the presence and/or the level of PSA proteins inmethods to monitor PSA protein overexpression (see, e.g., Stephan etal., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells(see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the193P1E1B polypeptides described herein can be utilized to generateantibodies for use in detecting 193P1E1B overexpression or themetastasis of prostate cells and cells of other cancers expressing thisgene.

Specifically, because metastases involves the movement of cancer cellsfrom an organ of origin (such as the lung or prostate gland etc.) to adifferent area of the body (such as a lymph node), assays which examinea biological sample for the presence of cells expressing 193P1E1Bpolynucleotides and/or polypeptides can be used to provide evidence ofmetastasis. For example, when a biological sample from tissue that doesnot normally contain 193P1E1B-expressing cells (lymph node) is found tocontain 193P1E1B-expressing cells such as the 193P1E1B expression seenin LAPC4 and LAPC9, xenografts isolated from lymph node and bonemetastasis, respectively, this finding is indicative of metastasis.

Alternatively 193P1E1B polynucleotides and/or polypeptides can be usedto provide evidence of cancer, for example, when cells in a biologicalsample that do not normally express 193P1E1B or express 193P1E1B at adifferent level are found to express 193P1E1B or have an increasedexpression of 193P1E1B (see, e.g., the 193P1E1B expression in thecancers listed in Table I and in patient samples etc. shown in theaccompanying Figures). In such assays, artisans may further wish togenerate supplementary evidence of metastasis by testing the biologicalsample for the presence of a second tissue restricted marker (inaddition to 193P1E1B) such as PSA, PSCA etc. (see, e.g., Alanen et al.,Pathol. Res. Pract. 192(3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants areemployed by skilled artisans for use in methods of monitoring PSA,193P1E1B polynucleotide fragments and polynucleotide variants are usedin an analogous manner. In particular, typical PSA polynucleotides usedin methods of monitoring PSA are probes or primers which consist offragments of the PSA cDNA sequence. Illustrating this, primers used toPCR amplify a PSA polynucleotide must include less than the whole PSAsequence to function in the polymerase chain reaction. In the context ofsuch PCR reactions, skilled artisans generally create a variety ofdifferent polynucleotide fragments that can be used as primers in orderto amplify different portions of a polynucleotide of interest or tooptimize amplification reactions (see, e.g., Caetano-Anolles, G.Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., MethodsMol. Biol. 98:121-154 (1998)). An additional illustration of the use ofsuch fragments is provided in the Example entitled “Expression analysisof 193P1E1B in normal tissues, and patient specimens,” where a 193P1E1Bpolynucleotide fragment is used as a probe to show the expression of193P1E1B RNAs in cancer cells. In addition, variant polynucleotidesequences are typically used as primers and probes for the correspondingmRNAs in PCR and Northern analyses (see, e.g., Sawai et al., FetalDiagn. Ther. November-December 1996 11 (6):407-13 and Current ProtocolsIn Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al.eds., 1995)). Polynucleotide fragments and variants are useful in thiscontext where they are capable of binding to a target polynucleotidesequence (e.g., a 193P1E1B polynucleotide shown in FIG. 2 or variantthereof) under conditions of high stringency.

Furthermore, PSA polypeptides which contain an epitope that can berecognized by an antibody or T cell that specifically binds to thatepitope are used in methods of monitoring PSA. 193P1E1B polypeptidefragments and polypeptide analogs or variants can also be used in ananalogous manner. This practice of using polypeptide fragments orpolypeptide variants to generate antibodies (such as anti-PSA antibodiesor T cells) is typical in the art with a wide variety of systems such asfusion proteins being used by practitioners (see, e.g., CurrentProtocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubelet al. eds., 1995). In this context, each epitope(s) functions toprovide the architecture with which an antibody or T cell is reactive.Typically, skilled artisans create a variety of different polypeptidefragments that can be used in order to generate immune responsesspecific for different portions of a polypeptide of interest (see, e.g.,U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it maybe preferable to utilize a polypeptide comprising one of the 193P1E1Bbiological motifs discussed herein or a motif-bearing subsequence whichis readily identified by one of skill in the art based on motifsavailable in the art. Polypeptide fragments, variants or analogs aretypically useful in this context as long as they comprise an epitopecapable of generating an antibody or T cell specific for a targetpolypeptide sequence (e.g. a 193P1E1B polypeptide shown in FIG. 3).

As shown herein, the 193P1E1B polynucleotides and polypeptides (as wellas the 193P1E1B polynucleotide probes and anti-193P1E1B antibodies or Tcells used to identify the presence of these molecules) exhibit specificproperties that make them useful in diagnosing cancers such as thoselisted in Table I. Diagnostic assays that measure the presence of193P1E1B gene products, in order to evaluate the presence or onset of adisease condition described herein, such as prostate cancer, are used toidentify patients for preventive measures or further monitoring, as hasbeen done so successfully with PSA. Moreover, these materials satisfy aneed in the art for molecules having similar or complementarycharacteristics to PSA in situations where, for example, a definitediagnosis of metastasis of prostatic origin cannot be made on the basisof a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract.192(3): 233-237 (1996)), and consequently, materials such as 193P1E1Bpolynucleotides and polypeptides (as well as the 193P1E1B polynucleotideprobes and anti-193P1E1B antibodies used to identify the presence ofthese molecules) need to be employed to confirm a metastases ofprostatic origin.

Finally, in addition to their use in diagnostic assays, the 193P1E1Bpolynucleotides disclosed herein have a number of other utilities suchas their use in the identification of oncogenetic associated chromosomalabnormalities in the chromosomal region to which the 193P1E1B gene maps(see the Example entitled “Chromosomal Mapping of 193P1E1B” below).Moreover, in addition to their use in diagnostic assays, the193P1E1B-related proteins and polynucleotides disclosed herein haveother utilities such as their use in the forensic analysis of tissues ofunknown origin (see, e.g., Takahama K Forensic Sci Int Jun. 28,1996;80(1-2): 63-9).

Additionally, 193P1E1B-related proteins or polynucleotides of theinvention can be used to treat a pathologic condition characterized bythe over-expression of 193P1E1B. For example, the amino acid or nucleicacid sequence of FIG. 2 or FIG. 3, or fragments of either, can be usedto generate an immune response to a 193P1E1B antigen. Antibodies orother molecules that react with 193P1E1B can be used to modulate thefunction of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 193P1E1B Protein Function

The invention includes various methods and compositions for inhibitingthe binding of 193P1E1B to its binding partner or its association withother protein(s) as well as methods for inhibiting 193P1E1B function.

XII.A.) Inhibition of 193P1E1B With Intracellular Antibodies

In one approach, a recombinant vector that encodes single chainantibodies that specifically bind to 193P1E1B are introduced into193P1E1B expressing cells via gene transfer technologies. Accordingly,the encoded single chain anti-193P1E1B antibody is expressedintracellularly, binds to 193P1E1B protein, and thereby inhibits itsfunction. Methods for engineering such intracellular single chainantibodies are well known. Such intracellular antibodies, also known as“intrabodies”, are specifically targeted to a particular compartmentwithin the cell, providing control over where the inhibitory activity ofthe treatment is focused. This technology has been successfully appliedin the art (for review, see Richardson and Marasco, 1995, TIBTECH vol.13). Intrabodies have been shown to virtually eliminate the expressionof otherwise abundant cell surface receptors (see, e.g., Richardson etal., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther.1: 332-337).

Single chain antibodies comprise the variable domains of the heavy andlight chain joined by a flexible linker polypeptide, and are expressedas a single polypeptide. Optionally, single chain antibodies areexpressed as a single chain variable region fragment joined to the lightchain constant region. Well-known intracellular trafficking signals areengineered into recombinant polynucleotide vectors encoding such singlechain antibodies in order to target precisely the intrabody to thedesired intracellular compartment. For example, intrabodies targeted tothe endoplasmic reticulum (ER) are engineered to incorporate a leaderpeptide and, optionally, a C-terminal ER retention signal, such as theKDEL amino acid motif. Intrabodies intended to exert activity in thenucleus are engineered to include a nuclear localization signal. Lipidmoieties are joined to intrabodies in order to tether the intrabody tothe cytosolic side of the plasma membrane. Intrabodies can also betargeted to exert function in the cytosol. For example, cytosolicintrabodies are used to sequester factors within the cytosol, therebypreventing them from being transported to their natural cellulardestination.

In one embodiment, intrabodies are used to capture 193P1E1B in thenucleus, thereby preventing its activity within the nucleus. Nucleartargeting signals are engineered into such 193P1E1B intrabodies in orderto achieve the desired targeting. Such 193P1E1B intrabodies are designedto bind specifically to a particular 193P1E1B domain. In anotherembodiment, cytosolic intrabodies that specifically bind to a 193P1E1Bprotein are used to prevent 193P1E1B from gaining access to the nucleus,thereby preventing it from exerting any biological activity within thenucleus (e.g., preventing 193P1E1B from forming transcription complexeswith other factors).

In order to specifically direct the expression of such intrabodies toparticular cells, the transcription of the intrabody is placed under theregulatory control of an appropriate tumor-specific promoter and/orenhancer. In order to target intrabody expression specifically toprostate, for example, the PSA promoter and/or promoter/enhancer can beutilized (See, for example, U.S. Pat. No. 5,919,652 issued Jul. 6,1999).

XII.B.) Inhibition of 193P1E1B with Recombinant Proteins

In another approach, recombinant molecules bind to 193P1E1B and therebyinhibit 193P1E1B function. For example, these recombinant moleculesprevent or inhibit 193P1E1B from accessing/binding to its bindingpartner(s) or associating with other protein(s). Such recombinantmolecules can, for example, contain the reactive part(s) of a 193P1E1Bspecific antibody molecule. In a particular embodiment, the 193P1E1Bbinding domain of a 193P1E1B binding partner is engineered into adimeric fusion protein, whereby the fusion protein comprises two193P1E1B ligand binding domains linked to the Fc portion of a human IgG,such as human IgG1. Such IgG portion can contain, for example, theC_(H)2 and C_(H)3 domains and the hinge region, but not the C_(H)1domain. Such dimeric fusion proteins are administered in soluble form topatients suffering from a cancer associated with the expression of193P1E1B, whereby the dimeric fusion protein specifically binds to193P1E1B and blocks 193P1E1B interaction with a binding partner. Suchdimeric fusion proteins are further combined into multimeric proteinsusing known antibody linking technologies.

XII.C.) Inhibition of 193P1E1B Transcription or Translation

The present invention also comprises various methods and compositionsfor inhibiting the transcription of the 193P1E1B gene. Similarly, theinvention also provides methods and compositions for inhibiting thetranslation of 193P1E1B mRNA into protein.

In one approach, a method of inhibiting the transcription of the193P1E1B gene comprises contacting the 193P1E1B gene with a 193P1E1Bantisense polynucleotide. In another approach, a method of inhibiting193P1E1B mRNA translation comprises contacting a 193P1E1B mRNA with anantisense polynucleotide. In another approach, a 193P1E1B specificribozyme is used to cleave a 193P1E1B message, thereby inhibitingtranslation. Such antisense and ribozyme based methods can also bedirected to the regulatory regions of the 193P1E1B gene, such as193P1E1B promoter and/or enhancer elements. Similarly, proteins capableof inhibiting a 193P1E1B gene transcription factor are used to inhibit193P1E1B mRNA transcription. The various polynucleotides andcompositions useful in the aforementioned methods have been describedabove. The use of antisense and ribozyme molecules to inhibittranscription and translation is well known in the art.

Other factors that inhibit the transcription of 193P1E1B by interferingwith 193P1E1B transcriptional activation are also useful to treatcancers expressing 193P1E1B. Similarly, factors that interfere with193P1E1B processing are useful to treat cancers that express 193P1E1B.Cancer treatment methods utilizing such factors are also within thescope of the invention.

XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies can be used to delivertherapeutic polynucleotide molecules to tumor cells synthesizing193P1E1B (i.e., antisense, ribozyme, polynucleotides encodingintrabodies and other 193P1E1B inhibitory molecules). A number of genetherapy approaches are known in the art. Recombinant vectors encoding193P1E1B antisense polynucleotides, ribozymes, factors capable ofinterfering with 193P1E1B transcription, and so forth, can be deliveredto target tumor cells using such gene therapy approaches.

The above therapeutic approaches can be combined with any one of a widevariety of surgical, chemotherapy or radiation therapy regimens. Thetherapeutic approaches of the invention can enable the use of reduceddosages of chemotherapy (or other therapies) and/or less frequentadministration, an advantage for all patients and particularly for thosethat do not tolerate the toxicity of the chemotherapeutic agent well.

The anti-tumor activity of a particular composition (e.g., antisense,ribozyme, intrabody), or a combination of such compositions, can beevaluated using various in vitro and in vivo assay systems. In vitroassays that evaluate therapeutic activity include cell growth assays,soft agar assays and other assays indicative of tumor promotingactivity, binding assays capable of determining the extent to which atherapeutic composition will inhibit the binding of 193P1E1B to abinding partner, etc.

In vivo, the effect of a 193P1E1B therapeutic composition can beevaluated in a suitable animal model. For example, xenogenic prostatecancer models can be used, wherein human prostate cancer explants orpassaged xenograft tissues are introduced into immune compromisedanimals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine3: 402-408). For example, PCT Patent Application WO98/16628 and U.S.Pat. No. 6,107,540 describe various xenograft models of human prostatecancer capable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy can be predicted usingassays that measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful inevaluating therapeutic compositions. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Therapeutic formulations can be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like. Apreferred formulation for intravenous injection comprises thetherapeutic composition in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. Therapeutic protein preparations can be lyophilized and stored assterile powders, preferably under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection.

Dosages and administration protocols for the treatment of cancers usingthe foregoing methods will vary with the method and the target cancer,and will generally depend on a number of other factors appreciated inthe art.

XIII.) Identification, Characterization and Use of Modulators of193P1E1B

Methods to Identify and Use Modulators

In one embodiment, screening is performed to identify modulators thatinduce or suppress a particular expression profile, suppress or inducespecific pathways, preferably generating the associated phenotypethereby. In another embodiment, having identified differentiallyexpressed genes important in a particular state; screens are performedto identify modulators that alter expression of individual genes, eitherincrease or decrease. In another embodiment, screening is performed toidentify modulators that alter a biological function of the expressionproduct of a differentially expressed gene. Again, having identified theimportance of a gene in a particular state, screens are performed toidentify agents that bind and/or modulate the biological activity of thegene product.

In addition, screens are done for genes that are induced in response toa candidate agent. After identifying a modulator (one that suppresses acancer expression pattern leading to a normal expression pattern, or amodulator of a cancer gene that leads to expression of the gene as innormal tissue) a screen is performed to identify genes that arespecifically modulated in response to the agent. Comparing expressionprofiles between normal tissue and agent-treated cancer tissue revealsgenes that are not expressed in normal tissue or cancer tissue, but areexpressed in agent treated tissue, and vice versa. These agent-specificsequences are identified and used by methods described herein for cancergenes or proteins. In particular these sequences and the proteins theyencode are used in marking or identifying agent-treated cells. Inaddition, antibodies are raised against the agent-induced proteins andused to target novel therapeutics to the treated cancer tissue sample.

Modulator-Related Identification and Screening Assays:

Gene Expression-Related Assays

Proteins, nucleic acids, and antibodies of the invention are used inscreening assays. The cancer-associated proteins, antibodies, nucleicacids, modified proteins and cells containing these sequences are usedin screening assays, such as evaluating the effect of drug candidates ona “gene expression profile,” expression profile of polypeptides oralteration of biological function. In one embodiment, the expressionprofiles are used, preferably in conjunction with high throughputscreening techniques to allow monitoring for expression profile genesafter treatment with a candidate agent (e.g., Davis, G F, et al., J BiolScreen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid,Genome Res 6:986-94, 1996).

The cancer proteins, antibodies, nucleic acids, modified proteins andcells containing the native or modified cancer proteins or genes areused in screening assays. That is, the present invention comprisesmethods for screening for compositions which modulate the cancerphenotype or a physiological function of a cancer protein of theinvention. This is done on a gene itself or by evaluating the effect ofdrug candidates on a “gene expression profile” or biological function.In one embodiment, expression profiles are used, preferably inconjunction with high throughput screening techniques to allowmonitoring after treatment with a candidate agent, see Zlokamik, supra.

A variety of assays are executed directed to the genes and proteins ofthe invention. Assays are run on an individual nucleic acid or proteinlevel. That is, having identified a particular gene as up regulated incancer, test compounds are screened for the ability to modulate geneexpression or for binding to the cancer protein of the invention.“Modulation” in this context includes an increase or a decrease in geneexpression. The preferred amount of modulation will depend on theoriginal change of the gene expression in normal versus tissueundergoing cancer, with changes of at least 10%, preferably 50%, morepreferably 100-300%, and in some embodiments 300-1000% or greater. Thus,if a gene exhibits a 4-fold increase in cancer tissue compared to normaltissue, a decrease of about four-fold is often desired; similarly, a10-fold decrease in cancer tissue compared to normal tissue a targetvalue of a 10-fold increase in expression by the test compound is oftendesired. Modulators that exacerbate the type of gene expression seen incancer are also useful, e.g., as an upregulated target in furtheranalyses.

The amount of gene expression is monitored using nucleic acid probes andthe quantification of gene expression levels, or, alternatively, a geneproduct itself is monitored, e.g., through the use of antibodies to thecancer protein and standard immunoassays. Proteomics and separationtechniques also allow for quantification of expression.

Expression Monitoring to Identify Compounds that Modify Gene Expression

In one embodiment, gene expression monitoring, i.e., an expressionprofile, is monitored simultaneously for a number of entities. Suchprofiles will typically involve one or more of the genes of FIG. 2. Inthis embodiment, e.g., cancer nucleic acid probes are attached tobiochips to detect and quantify cancer sequences in a particular cell.Alternatively, PCR can be used. Thus, a series, e.g., wells of amicrotiter plate, can be used with dispensed primers in desired wells. APCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify theexpression of one or more cancer-associated sequences, e.g., apolynucleotide sequence set out in FIG. 2. Generally, a test modulatoris added to the cells prior to analysis. Moreover, screens are alsoprovided to identify agents that modulate cancer, modulate cancerproteins of the invention, bind to a cancer protein of the invention, orinterfere with the binding of a cancer protein of the invention and anantibody or other binding partner.

In one embodiment, high throughput screening methods involve providing alibrary containing a large number of potential therapeutic compounds(candidate compounds). Such “combinatorial chemical libraries” are thenscreened in one or more assays to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve asconventional “lead compounds,” as compounds for screening, or astherapeutics.

In certain embodiments, combinatorial libraries of potential modulatorsare screened for an ability to bind to a cancer polypeptide or tomodulate activity. Conventionally, new chemical entities with usefulproperties are generated by identifying a chemical compound (called a“lead compound”) with some desirable property or activity, e.g.,inhibiting activity, creating variants of the lead compound, andevaluating the property and activity of those variant compounds. Often,high throughput screening (HTS) methods are employed for such ananalysis.

As noted above, gene expression monitoring is conveniently used to testcandidate modulators (e.g., protein, nucleic acid or small molecule).After the candidate agent has been added and the cells allowed toincubate for a period, the sample containing a target sequence to beanalyzed is, e.g., added to a biochip.

If required, the target sequence is prepared using known techniques. Forexample, a sample is treated to lyse the cells, using known lysisbuffers, electroporation, etc., with purification and/or amplificationsuch as PCR performed as appropriate. For example, an in vitrotranscription with labels covalently attached to the nucleotides isperformed. Generally, the nucleic acids are labeled with biotin-FITC orPE, or with cy3 or cy5.

The target sequence can be labeled with, e.g., a fluorescent, achemiluminescent, a chemical, or a radioactive signal, to provide ameans of detecting the target sequence's specific binding to a probe.The label also can be an enzyme, such as alkaline phosphatase orhorseradish peroxidase, which when provided with an appropriatesubstrate produces a product that is detected. Alternatively, the labelis a labeled compound or small molecule, such as an enzyme inhibitor,that binds but is not catalyzed or altered by the enzyme. The label alsocan be a moiety or compound, such as, an epitope tag or biotin whichspecifically binds to streptavidin. For the example of biotin, thestreptavidin is labeled as described above, thereby, providing adetectable signal for the bound target sequence. Unbound labeledstreptavidin is typically removed prior to analysis.

As will be appreciated by those in the art, these assays can be directhybridization assays or can comprise “sandwich assays”, which includethe use of multiple probes, as is generally outlined in U.S. Pat. Nos.5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670;5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118;5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, thetarget nucleic acid is prepared as outlined above, and then added to thebiochip comprising a plurality of nucleic acid probes, under conditionsthat allow the formation of a hybridization complex.

A variety of hybridization conditions are used in the present invention,including high, moderate and low stringency conditions as outlinedabove. The assays are generally run under stringency conditions whichallow formation of the label probe hybridization complex only in thepresence of target. Stringency can be controlled by altering a stepparameter that is a thermodynamic variable, including, but not limitedto, temperature, formamide concentration, salt concentration, chaotropicsalt concentration pH, organic solvent concentration, etc. Theseparameters may also be used to control non-specific binding, as isgenerally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirableto perform certain steps at higher stringency conditions to reducenon-specific binding.

The reactions outlined herein can be accomplished in a variety of ways.Components of the reaction can be added simultaneously, or sequentially,in different orders, with preferred embodiments outlined below. Inaddition, the reaction may include a variety of other reagents. Theseinclude salts, buffers, neutral proteins, e.g. albumin, detergents, etc.which can be used to facilitate optimal hybridization and detection,and/or reduce nonspecific or background interactions. Reagents thatotherwise improve the efficiency of the assay, such as proteaseinhibitors, nuclease inhibitors, anti-microbial agents, etc., may alsobe used as appropriate, depending on the sample preparation methods andpurity of the target. The assay data are analyzed to determine theexpression levels of individual genes, and changes in expression levelsas between states, forming a gene expression profile.

Biological Activity-Related Assays

The invention provides methods identify or screen for a compound thatmodulates the activity of a cancer-related gene or protein of theinvention. The methods comprise adding a test compound, as definedabove, to a cell comprising a cancer protein of the invention. The cellscontain a recombinant nucleic acid that encodes a cancer protein of theinvention. In another embodiment, a library of candidate agents istested on a plurality of cells.

In one aspect, the assays are evaluated in the presence or absence orprevious or subsequent exposure of physiological signals, e.g. hormones,antibodies, peptides, antigens, cytokines, growth factors, actionpotentials, pharmacological agents including chemotherapeutics,radiation, carcinogenics, or other cells (i.e., cell-cell contacts). Inanother example, the determinations are made at different stages of thecell cycle process. In this way, compounds that modulate genes orproteins of the invention are identified. Compounds with pharmacologicalactivity are able to enhance or interfere with the activity of thecancer protein of the invention. Once identified, similar structures areevaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (e.g., inhibiting) cancer celldivision is provided; the method comprises administration of a cancermodulator. In another embodiment, a method of modulating (e.g.,inhibiting) cancer is provided; the method comprises administration of acancer modulator. In a further embodiment, methods of treating cells orindividuals with cancer are provided; the method comprisesadministration of a cancer modulator.

In one embodiment, a method for modulating the status of a cell thatexpresses a gene of the invention is provided. As used herein statuscomprises such art-accepted parameters such as growth, proliferation,survival, function, apoptosis, senescence, location, enzymatic activity,signal transduction, etc. of a cell. In one embodiment, a cancerinhibitor is an antibody as discussed above. In another embodiment, thecancer inhibitor is an antisense molecule. A variety of cell growth,proliferation, and metastasis assays are known to those of skill in theart, as described herein.

High Throughput Screening to Identify Modulators

The assays to identify suitable modulators are amenable to highthroughput screening. Preferred assays thus detect enhancement orinhibition of cancer gene transcription, inhibition or enhancement ofpolypeptide expression, and inhibition or enhancement of polypeptideactivity.

In one embodiment, modulators evaluated in high throughput screeningmethods are proteins, often naturally occurring proteins or fragments ofnaturally occurring proteins. Thus, e.g., cellular extracts containingproteins, or random or directed digests of proteinaceous cellularextracts, are used. In this way, libraries of proteins are made forscreening in the methods of the invention. Particularly preferred inthis embodiment are libraries of bacterial, fungal, viral, and mammalianproteins, with the latter being preferred, and human proteins beingespecially preferred. Particularly useful test compound will be directedto the class of proteins to which the target belongs, e.g., substratesfor enzymes, or ligands and receptors.

Use of Soft Agar Growth and Colony Formation to Identify andCharacterize Modulators

Normal cells require a solid substrate to attach and grow. When cellsare transformed, they lose this phenotype and grow detached from thesubstrate. For example, transformed cells can grow in stirred suspensionculture or suspended in semi-solid media, such as semi-solid or softagar. The transformed cells, when transfected with tumor suppressorgenes, can regenerate normal phenotype and once again require a solidsubstrate to attach to and grow. Soft agar growth or colony formation inassays are used to identify modulators of cancer sequences, which whenexpressed in host cells, inhibit abnormal cellular proliferation andtransformation. A modulator reduces or eliminates the host cells'ability to grow suspended in solid or semisolid media, such as agar.

Techniques for soft agar growth or colony formation in suspension assaysare described in Freshney, Culture of Animal Cells a Manual of BasicTechnique (3rd ed., 1994). See also, the methods section of Garkavtsevet al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation toIdentify and Characterize Modulators

Normal cells typically grow in a flat and organized pattern in cellculture until they touch other cells. When the cells touch one another,they are contact inhibited and stop growing. Transformed cells, however,are not contact inhibited and continue to grow to high densities indisorganized foci. Thus, transformed cells grow to a higher saturationdensity than corresponding normal cells. This is detectedmorphologically by the formation of a disoriented monolayer of cells orcells in foci. Alternatively, labeling index with (³H)-thymidine atsaturation density is used to measure density limitation of growth,similarly an MTT or Alamar blue assay will reveal proliferation capacityof cells and the the ability of modulators to affect same. See Freshney(1994), supra. Transformed cells, when transfected with tumor suppressorgenes, can regenerate a normal phenotype and become contact inhibitedand would grow to a lower density.

In this assay, labeling index with ³H)-thymidine at saturation densityis a preferred method of measuring density limitation of growth.Transformed host cells are transfected with a cancer-associated sequenceand are grown for 24 hours at saturation density in non-limiting mediumconditions. The percentage of cells labeling with (³H)-thymidine isdetermined by incorporated cpm.

Contact independent growth is used to identify modulators of cancersequences, which had led to abnormal cellular proliferation andtransformation. A modulator reduces or eliminates contact independentgrowth, and returns the cells to a normal phenotype.

Evaluation of Growth Factor or Serum Dependence to Identify andCharacterize Modulators

Transformed cells have lower serum dependence than their normalcounterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966);Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This isin part due to release of various growth factors by the transformedcells. The degree of growth factor or serum dependence of transformedhost cells can be compared with that of control. For example, growthfactor or serum dependence of a cell is monitored in methods to identifyand characterize compounds that modulate cancer-associated sequences ofthe invention.

Use of Tumor-specific Marker Levels to Identify and CharacterizeModulators

Tumor cells release an increased amount of certain factors (hereinafter“tumor specific markers”) than their normal counterparts. For example,plasminogen activator (PA) is released from human glioma at a higherlevel than from normal brain cells (see, e.g., Gullino, Angiogenesis,Tumor Vascularization, and Potential Interference with Tumor Growth, inBiological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)).Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher levelin tumor cells than their normal counterparts. See, e.g., Folkman,Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF isreleased from endothelial tumors (Ensoli, B et al).

Various techniques which measure the release of these factors aredescribed in Freshney (1994), supra. Also, see, Unkless et al., J. Biol.Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem.251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980);Gullino, Angiogenesis, Tumor Vascularization, and Potential Interferencewith Tumor Growth, in Biological Responses in Cancer, pp. 178-184(Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). Forexample, tumor specific marker levels are monitored in methods toidentify and characterize compounds that modulate cancer-associatedsequences of the invention.

Invasiveness into Matrigel to Identify and Characterize Modulators

The degree of invasiveness into Matrigel or an extracellular matrixconstituent can be used as an assay to identify and characterizecompounds that modulate cancer associated sequences. Tumor cells exhibita positive correlation between malignancy and invasiveness of cells intoMatrigel or some other extracellular matrix constituent. In this assay,tumorigenic cells are typically used as host cells. Expression of atumor suppressor gene in these host cells would decrease invasiveness ofthe host cells. Techniques described in Cancer Res. 1999; 59:6010;Freshney (1994), supra, can be used. Briefly, the level of invasion ofhost cells is measured by using filters coated with Matrigel or someother extracellular matrix constituent. Penetration into the gel, orthrough to the distal side of the filter, is rated as invasiveness, andrated histologically by number of cells and distance moved, or byprelabeling the cells with ¹²⁵1 and counting the radioactivity on thedistal side of the filter or bottom of the dish. See, e.g., Freshney(1984), supra.

Evaluation of Tumor Growth In Vivo to Identify and CharacterizeModulators

Effects of cancer-associated sequences on cell growth are tested intransgenic or immune-suppressed organisms. Transgenic organisms areprepared in a variety of art-accepted ways. For example, knock-outtransgenic organisms, e.g., mammals such as mice, are made, in which acancer gene is disrupted or in which a cancer gene is inserted.Knock-out transgenic mice are made by insertion of a marker gene orother heterologous gene into the endogenous cancer gene site in themouse genome via homologous recombination. Such mice can also be made bysubstituting the endogenous cancer gene with a mutated version of thecancer gene, or by mutating the endogenous cancer gene, e.g., byexposure to carcinogens.

To prepare transgenic chimeric animals, e.g., mice, a DNA construct isintroduced into the nuclei of embryonic stem cells. Cells containing thenewly engineered genetic lesion are injected into a host mouse embryo,which is re-implanted into a recipient female. Some of these embryosdevelop into chimeric mice that possess germ cells some of which arederived from the mutant cell line. Therefore, by breeding the chimericmice it is possible to obtain a new line of mice containing theintroduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288(1989)). Chimeric mice can be derived according to U.S. Pat. No.6,365,797, issued Apr. 2, 2002; U.S. Pat. No. 6,107,540 issued Aug. 22,2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual,Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,D.C., (1987).

Alternatively, various immune-suppressed or immune-deficient hostanimals can be used. For example, a genetically athymic “nude” mouse(see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), aSCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g.,Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer41:52 (1980)) can be used as a host. Transplantable tumor cells(typically about 10⁶ cells) injected into isogenic hosts produceinvasive tumors in a high proportion of cases, while normal cells ofsimilar origin will not. In hosts which developed invasive tumors, cellsexpressing cancer-associated sequences are injected subcutaneously ororthotopically. Mice are then separated into groups, including controlgroups and treated experimental groups) e.g. treated with a modulator).After a suitable length of time, preferably 4-8 weeks, tumor growth ismeasured (e.g., by volume or by its two largest dimensions, or weight)and compared to the control. Tumors that have statistically significantreduction (using, e.g., Student's T test) are said to have inhibitedgrowth.

In Vitro Assays to Identify and Characterize Modulators

Assays to identify compounds with modulating activity can be performedin vitro. For example, a cancer polypeptide is first contacted with apotential modulator and incubated for a suitable amount of time, e.g.,from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levelsare determined in vitro by measuring the level of protein or mRNA. Thelevel of protein is measured using immunoassays such as Westernblotting, ELISA and the like with an antibody that selectively binds tothe cancer polypeptide or a fragment thereof. For measurement of mRNA,amplification, e.g., using PCR, LCR, or hybridization assays, e. g.,Northern hybridization, RNAse protection, dot blotting, are preferred.The level of protein or mRNA is detected using directly or indirectlylabeled detection agents, e.g., fluorescently or radioactively labelednucleic acids, radioactively or enzymatically labeled antibodies, andthe like, as described herein.

Alternatively, a reporter gene system can be devised using a cancerprotein promoter operably linked to a reporter gene such as luciferase,green fluorescent protein, CAT, or P-gal. The reporter construct istypically transfected into a cell. After treatment with a potentialmodulator, the amount of reporter gene transcription, translation, oractivity is measured according to standard techniques known to those ofskill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr.Opin. Biotechnol. 1998: 9:624).

As outlined above, in vitro screens are done on individual genes andgene products. That is, having identified a particular differentiallyexpressed gene as important in a particular state, screening ofmodulators of the expression of the gene or the gene product itself isperformed.

In one embodiment, screening for modulators of expression of specificgene(s) is performed. Typically, the expression of only one or a fewgenes is evaluated. In another embodiment, screens are designed to firstfind compounds that bind to differentially expressed proteins. Thesecompounds are then evaluated for the ability to modulate differentiallyexpressed activity. Moreover, once initial candidate compounds areidentified, variants can be further screened to better evaluatestructure activity relationships.

Binding Assays to Identify and Characterize Modulators

In binding assays in accordance with the invention, a purified orisolated gene product of the invention is generally used. For example,antibodies are generated to a protein of the invention, and immunoassaysare run to determine the amount and/or location of protein.Alternatively, cells comprising the cancer proteins are used in theassays.

Thus, the methods comprise combining a cancer protein of the inventionand a candidate compound such as a ligand, and determining the bindingof the compound to the cancer protein of the invention. Preferredembodiments utilize the human cancer protein; animal models of humandisease of can also be developed and used. Also, other analogousmammalian proteins also can be used as appreciated by those of skill inthe art. Moreover, in some embodiments variant or derivative cancerproteins are used.

Generally, the cancer protein of the invention, or the ligand, isnon-diffusibly bound to an insoluble support. The support can, e.g., beone having isolated sample receiving areas (a microtiter plate, anarray, etc.). The insoluble supports can be made of any composition towhich the compositions can be bound, is readily separated from solublematerial, and is otherwise compatible with the overall method ofscreening. The surface of such supports can be solid or porous and ofany convenient shape.

Examples of suitable insoluble supports include microtiter plates,arrays, membranes and beads. These are typically made of glass, plastic(e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon™,etc. Microtiter plates and arrays are especially convenient because alarge number of assays can be carried out simultaneously, using smallamounts of reagents and samples. The particular manner of binding of thecomposition to the support is not crucial so long as it is compatiblewith the reagents and overall methods of the invention, maintains theactivity of the composition and is nondiffusable. Preferred methods ofbinding include the use of antibodies which do not sterically blockeither the ligand binding site or activation sequence when attaching theprotein to the support, direct binding to “sticky” or ionic supports,chemical crosslinking, the synthesis of the protein or agent on thesurface, etc. Following binding of the protein or ligand/binding agentto the support, excess unbound material is removed by washing. Thesample receiving areas may then be blocked through incubation withbovine serum albumin (BSA), casein or other innocuous protein or othermoiety.

Once a cancer protein of the invention is bound to the support, and atest compound is added to the assay. Alternatively, the candidatebinding agent is bound to the support and the cancer protein of theinvention is then added. Binding agents include specific antibodies,non-natural binding agents identified in screens of chemical libraries,peptide analogs, etc.

Of particular interest are assays to identify agents that have a lowtoxicity for human cells. A wide variety of assays can be used for thispurpose, including proliferation assays, cAMP assays, labeled in vitroprotein-protein binding assays, electrophoretic mobility shift assays,immunoassays for protein binding, functional assays (phosphorylationassays, etc.) and the like.

A determination of binding of the test compound (ligand, binding agent,modulator, etc.) to a cancer protein of the invention can be done in anumber of ways. The test compound can be labeled, and binding determineddirectly, e.g., by attaching all or a portion of the cancer protein ofthe invention to a solid support, adding a labeled candidate compound(e.g., a fluorescent label), washing off excess reagent, and determiningwhether the label is present on the solid support. Various blocking andwashing steps can be utilized as appropriate.

In certain embodiments, only one of the components is labeled, e.g., aprotein of the invention or ligands labeled. Alternatively, more thanone component is labeled with different labels, e.g., I¹²⁵, for theproteins and a fluorophor for the compound. Proximity reagents, e.g.,quenching or energy transfer reagents are also useful.

Competitive Binding to Identify and Characterize Modulators

In one embodiment, the binding of the “test compound” is determined bycompetitive binding assay with a “competitor.” The competitor is abinding moiety that binds to the target molecule (e.g., a cancer proteinof the invention). Competitors include compounds such as antibodies,peptides, binding partners, ligands, etc. Under certain circumstances,the competitive binding between the test compound and the competitordisplaces the test compound. In one embodiment, the test compound islabeled. Either the test compound, the competitor, or both, is added tothe protein for a time sufficient to allow binding. Incubations areperformed at a temperature that facilitates optimal activity, typicallybetween four and 40° C. Incubation periods are typically optimized,e.g., to facilitate rapid high throughput screening; typically betweenzero and one hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In one embodiment, the competitor is added first, followed by the testcompound. Displacement of the competitor is an indication that the testcompound is binding to the cancer protein and thus is capable of bindingto, and potentially modulating, the activity of the cancer protein. Inthis embodiment, either component can be labeled. Thus, e.g., if thecompetitor is labeled, the presence of label in the post-test compoundwash solution indicates displacement by the test compound.Alternatively, if the test compound is labeled, the presence of thelabel on the support indicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor indicates that the test compound binds to thecancer protein with higher affinity than the competitor. Thus, if thetest compound is labeled, the presence of the label on the support,coupled with a lack of competitor binding, indicates that the testcompound binds to and thus potentially modulates the cancer protein ofthe invention.

Accordingly, the competitive binding methods comprise differentialscreening to identity agents that are capable of modulating the activityof the cancer proteins of the invention. In this embodiment, the methodscomprise combining a cancer protein and a competitor in a first sample.A second sample comprises a test compound, the cancer protein, and acompetitor. The binding of the competitor is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to the cancerprotein and potentially modulating its activity. That is, if the bindingof the competitor is different in the second sample relative to thefirst sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drugcandidates that bind to the native cancer protein, but cannot bind tomodified cancer proteins. For example the structure of the cancerprotein is modeled and used in rational drug design to synthesize agentsthat interact with that site, agents which generally do not bind tosite-modified proteins. Moreover, such drug candidates that affect theactivity of a native cancer protein are also identified by screeningdrugs for the ability to either enhance or reduce the activity of suchproteins.

Positive controls and negative controls can be used in the assays.Preferably control and test samples are performed in at least triplicateto obtain statistically significant results. Incubation of all samplesoccurs for a time sufficient to allow for the binding of the agent tothe protein. Following incubation, samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples can be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents can be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc. which are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,can be used. The mixture of components is added in an order thatprovides for the requisite binding.

Use of Polynucleotides to Down-Regulate or Inhibit a Protein of theInvention.

Polynucleotide modulators of cancer can be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand-binding molecule, as described in WO 91/04753. Suitableligand-binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell. Alternatively, a polynucleotide modulator ofcancer can be introduced into a cell containing the target nucleic acidsequence, e.g., by formation of a polynucleotide-lipid complex, asdescribed in WO 90/10448. It is understood that the use of antisensemolecules or knock out and knock in models may also be used in screeningassays as discussed above, in addition to methods of treatment.

Inhibitory and Antisense Nucleotides

In certain embodiments, the activity of a cancer-associated protein isdown-regulated, or entirely inhibited, by the use of antisensepolynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleicacid complementary to, and which can preferably hybridize specificallyto, a coding mRNA nucleic acid sequence, e.g., a cancer protein of theinvention, mRNA, or a subsequence thereof. Binding of the antisensepolynucleotide to the mRNA reduces the translation and/or stability ofthe mRNA.

In the context of this invention, antisense polynucleotides can comprisenaturally occurring nucleotides, or synthetic species formed fromnaturally occurring subunits or their close homologs. Antisensepolynucleotides may also have altered sugar moieties or inter-sugarlinkages. Exemplary among these are the phosphorothioate and othersulfur containing species which are known for use in the art. Analogsare comprised by this invention so long as they function effectively tohybridize with nucleotides of the invention. See, e.g., IsisPharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.

Such antisense polynucleotides can readily be synthesized usingrecombinant means, or can be synthesized in vitro. Equipment for suchsynthesis is sold by several vendors, including Applied Biosystems. Thepreparation of other oligonucleotides such as phosphorothioates andalkylated derivatives is also well known to those of skill in the art.

Antisense molecules as used herein include antisense or senseoligonudeotides. Sense oligonucleotides can, e.g., be employed to blocktranscription by binding to the anti-sense strand. The antisense andsense oligonucleotide comprise a single stranded nucleic acid sequence(either RNA or DNA) capable of binding to target mRNA (sense) or DNA(antisense) sequences for cancer molecules. Antisense or senseoligonucleotides, according to the present invention, comprise afragment generally at least about 12 nucleotides, preferably from about12 to 30 nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van derKrol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used totarget and inhibit transcription of cancer-associated nucleotidesequences. A ribozyme is an RNA molecule that catalytically cleavesother RNA molecules. Different kinds of ribozymes have been described,including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. inPharmacology 25: 289-317 (1994) for a general review of the propertiesof different ribozymes).

The general features of hairpin ribozymes are described, e.g., in Hampelet al., Nucl. Acids Res. 18:299-304 (1990); European Patent PublicationNo. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are wellknown to those of skill in the art (see, e.g., WO 94/26877; Ojwang etal., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al.,Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci.USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120(1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

In one embodiment, a test compound is administered to a population ofcancer cells, which have an associated cancer expression profile. By“administration” or “contacting” herein is meant that the modulator isadded to the cells in such a manner as to allow the modulator to actupon the cell, whether by uptake and intracellular action, or by actionat the cell surface. In some embodiments, a nucleic acid encoding aproteinaceous agent (i.e., a peptide) is put into a viral construct suchas an adenoviral or retroviral construct, and added to the cell, suchthat expression of the peptide agent is accomplished, e.g., PCTUS97/01019. Regulatable gene therapy systems can also be used. Once themodulator has been administered to the cells, the cells are washed ifdesired and are allowed to incubate under preferably physiologicalconditions for some period. The cells are then harvested and a new geneexpression profile is generated. Thus, e.g., cancer tissue is screenedfor agents that modulate, e.g., induce or suppress, the cancerphenotype. A change in at least one gene, preferably many, of theexpression profile indicates that the agent has an effect on canceractivity. Similarly, altering a biological function or a signalingpathway is indicative of modulator activity. By defining such asignature for the cancer phenotype, screens for new drugs that alter thephenotype are devised. With this approach, the drug target need not beknown and need not be represented in the original gene/proteinexpression screening platform, nor does the level of transcript for thetarget protein need to change. The modulator inhibiting function willserve as a surrogate marker

As outlined above, screens are done to assess genes or gene products.That is, having identified a particular differentially expressed gene asimportant in a particular state, screening of modulators of either theexpression of the gene or the gene product itself is performed.

Use of Modulators to Affect Peptides of the Invention

Measurements of cancer polypeptide activity, or of the cancer phenotypeare performed using a variety of assays. For example, the effects ofmodulators upon the function of a cancer polypeptide(s) are measured byexamining parameters described above. A physiological change thataffects activity is used to assess the influence of a test compound onthe polypeptides of this invention. When the functional outcomes aredetermined using intact cells or animals, a variety of effects can beassesses such as, in the case of a cancer associated with solid tumors,tumor growth, tumor metastasis, neovascularization, hormone release,transcriptional changes to both known and uncharacterized geneticmarkers (e.g., by Northern blots), changes in cell metabolism such ascell growth or pH changes, and changes in intracellular secondmessengers such as cGNIP.

Methods of Identifying Characterizing Cancer-associated Sequences

Expression of various gene sequences is correlated with cancer.Accordingly, disorders based on mutant or variant cancer genes aredetermined. In one embodiment, the invention provides methods foridentifying cells containing variant cancer genes, e.g., determining thepresence of, all or part, the sequence of at least one endogenous cancergene in a cell. This is accomplished using any number of sequencingtechniques. The invention comprises methods of identifying the cancergenotype of an individual, e.g., determining all or part of the sequenceof at least one gene of the invention in the individual. This isgenerally done in at least one tissue of the individual, e.g., a tissueset forth in Table I, and may include the evaluation of a number oftissues or different samples of the same tissue. The method may includecomparing the sequence of the sequenced gene to a known cancer gene,i.e., a wild-type gene to determine the presence of family members,homologies, mutations or variants. The sequence of all or part of thegene can then be compared to the sequence of a known cancer gene todetermine if any differences exist. This is done using any number ofknown homology programs, such as BLAST, Besffit, etc. The presence of adifference in the sequence between the cancer gene of the patient andthe known cancer gene correlates with a disease state or a propensityfor a disease state, as outlined herein.

In a preferred embodiment, the cancer genes are used as probes todetermine the number of copies of the cancer gene in the genome. Thecancer genes are used as probes to determine the chromosomallocalization of the cancer genes. Information such as chromosomallocalization finds use in providing a diagnosis or prognosis inparticular when chromosomal abnormalities such as translocations, andthe like are identified in the cancer gene locus.

XIV.) Kits/Articles of Manufacture

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. Such probe can be an antibody orpolynucleotide specific for a FIG. 2-related protein or a FIG. 2 gene ormessage, respectively. Where the method utilizes nucleic acidhybridization to detect the target nucleic acid, the kit can also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradioisotope label. The kit can include all or part of the amino acidsequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acidmolecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use.

A label can be present on the container to indicate that the compositionis used for a specific therapy or non-therapeutic application, such as adiagnostic or laboratory application, and can also indicate directionsfor either in vivo or in viro use, such as those described herein.Directions and or other information can also be included on an insert(s)or label(s) which is included with or on the kit

The terms “kit” and “article of manufacture” can be used as synonyms.

In another embodiment of the invention, an article(s) of manufacturecontaining compositions, such as amino acid sequence(s), smallmolecule(s), nucleic acid sequence(s), and/or antibody(s), e.g.,materials useful for the diagnosis, prognosis, prophylaxis and/ortreatment of neoplasias of tissues such as those set forth in Table I isprovided. The article of manufacture typically comprises at least onecontainer and at least one label. Suitable containers include, forexample, bottles, vials, syringes, and test tubes. The containers can beformed from a variety of materials such as glass or plastic. Thecontainer can hold amino acid sequence(s), small molecule(s), nucleicacid sequence(s), and/or antibody(s), in one embodiment the containerholds a polynucleotide for use in examining the mRNA expression profileof a cell, together with reagents used for this purpose.

The container can alternatively hold a composition which is effectivefor treating, diagnosis, prognosing or prophylaxing a condition and canhave a sterile access port (for example the container can be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The active agents in the composition canbe an antibody capable of specifically binding 193P1E1B and modulatingthe function of 193P1E1B.

The label can be on or associated with the container. A label a can beon a container when letters, numbers or other characters forming thelabel are molded or etched into the container itself; a label can beassociated with a container when it is present within a receptacle orcarrier that also holds the container, e.g., as a package insert. Thelabel can indicate that the composition is used for diagnosing,treating, prophylaxing or prognosing a condition, such as a neoplasia ofa issue set forth in Table I. The article of manufacture can furthercomprise a second container comprising a pharmaceutically-acceptablebuffer, such as phosphate-buffered saline, Ringer's solutionand/ordextrose solution. It can further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, stirrers, needles, syringes, and/or packageinserts with indications and/or instructions for use.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples that follow, none of which are intendedto limit the scope of the invention.

Example 1 SSH-Generated Isolation of cDNA Fragment of the 193P1E1B Gene

To isolate genes that are over-expressed in prostate cancer we used theSuppression Subtractive Hybridization (SSH) procedure using cDNA derivedfrom prostate cancer xenograft tissues. LAPC-9AD xenograft was obtainedfrom Dr. Charles Sawyers (UCLA) and was generated as described (Klein etal., 1997, Nature Med. 3:402-408; Craft et al., 1999, Cancer Res.59:5030-5036). LAPC-9AD² was generated from LAPC-9AD xenograft bygrowing LAPC-9AD xenograft tissues within a piece of human boneimplanted in SCID mice. Tumors were then harvested and subsequentlypassaged subcutaneously into other SCID animals to generate LAPC-9AD².

The 193P1E1B SSH cDNA sequence was derived from a subtraction consistingof a prostate cancer xenograft LAPC-9AD² minus prostate cancer xenograftLAPC-9AD. By RT-PCR, the 193P1E1B cDNA was identified as highlyexpressed in the prostate cancer xenograft pool (LAPC4-AD, LAPC4-AI,LAPC9-AD, LAPC9-AI), bladder cancer pool, kidney cancer pool, coloncancer pool, lung cancer pool, ovary cancer pool, breast cancer pool,metastasis cancer pool, pancreas cancer pool, with low expressionobserved in the prostate cancer pool, and no expression observed invital pool 1 (kidney, liver, lung), and in vital pool 2 (stomach, colon,pancreas) (FIG. 14).

The 193P1E1B SSH cDNA of 227 bp is listed in FIG. 1. The full length193P1E1B cDNAs and ORFs are described in FIG. 2 with the proteinsequences listed in FIG. 3. 193P1E1B v.1, v.2, v.3, v.4, v.5, v.6, v.7,v.8, v.11, v.12 and v.13 are novel proteins and have not been previouslydescribed. 193P1E1B v.9 shows 99% identity to a hypothetical protein,MGC4832. 193P1E1B v.10 shows 100% identity to a novel unnamed proteinBAC03484.1.

Materials and Methods

RNA Isolation:

Tumor tissues were homogenized in Trizol reagent (Life Technologies,Gibco BRL) using 10 ml/g tissue or 10 ml/10⁸ cells to isolate total RNA.Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Miniand Midi kits. Total and mRNA were quantified by spectrophotometricanalysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer): 5′TTTTGATCAAGCTT₃₀3′ (SEQ ID NO: 52)Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′ (SEQ ID NO:53) 3′GGCCCGTCCTAG5′ (SEQ ID NO: 54) Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′ (SEQ ID NO: 55)3′CGGCTCCTAG5′ (SEQ ID NO: 56) PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′(SEQ ID NO: 57) Nested primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGA3′ (SEQ IDNO: 58) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGA3′ (SEQ ID NO: 59)

Suppression Subtractive Hybridization (SSH) was used to identify cDNAscorresponding to genes that may be differentially expressed in prostatecancer. The SSH reaction utilized cDNA from prostate cancer xenograftLAPC-9AD². The gene 193P1E1B was derived from a prostate cancerxenograft LAPC-9AD² minus prostate cancer xenograft LAPC-9AD tissues.The SSH DNA sequence (FIG. 1) was identified.

The cDNA derived from prostate cancer xenograft LAPC-9AD tissue was usedas the source of the “driver” cDNA, while the cDNA from prostate cancerxenograft LAPC-9AD² was used as the source of the tester cDNA. Doublestranded cDNAs corresponding to tester and driver cDNAs were synthesizedfrom 2 μg of poly(A)⁺ RNA isolated from the relevant tissue, asdescribed above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1ng of oligonucleotide DPNCDN as primer. First- and second-strandsynthesis were carried out as described in the Kit's user manualprotocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). Theresulting cDNA was digested with Dpn II for 3 hrs at 37° C. DigestedcDNA was extracted with phenol/chloroform (1:1) and ethanolprecipitated.

Tester cDNA was generated by diluting 1 p of Dpn II digested cDNA fromthe relevant tissue source (see above) (400 ng) in 5 μl of water. Thediluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 andAdaptor 2 (10 μM), in separate ligation reactions, in a total volume of10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH).Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C.for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Researchthermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10 x reaction buffer (CLONTECH) and0.5 μl 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volumeof 25 μl..PCR 1 was conducted using the following conditions: 75° C. for5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR productswere analyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dBest, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNAs can be generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturer's protocol was used which included anincubation for 50 min at 42° C. with reverse transcriptase followed byRNAse H treatment at 37° C. for 20 min. After completing the reaction,the volume can be increased to 200 μl with water prior to normalization.First strand cDNAs from 16 different normal human tissues can beobtained from Clontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO:60) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 61) to amplifyβ-acon. First strand cDNA (5 μl) were amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH8.3) and 1× Klentaq DNApolymerase (Clontech). Five μl of the PCR reaction can be removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research thermal cycler under the followingconditions: Initial denaturation can be at 94° C. for 15 sec, followedby a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C.for 5 sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization can be required to achieve equal bandintensities in all tissues after 22 cycles of PCR.

To determine expression levels of the 193P1E1B gene, 5 μl of normalizedfirst strand cDNA were analyzed by PCR using 26, and 30 cycles ofamplification. Semi-quantitative expression analysis can be achieved bycomparing the PCR products at cycle numbers that give light bandintensities.

A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCRexpression analysis was performed on first strand cDNA generated usingpools of tissues from multiple samples. The cDNA samples were shown tobe normalized using beta-actin PCR. Strong expression of 193P1E1B wasobserved in prostate cancer xenograft pool, bladder cancer pool, kidneycancer pool, colon cancer pool, lung cancer pool, ovary cancer pool,breast cancer pool, and metastasis pool. Low expression was observed inprostate cancer pool, but no expression was detected in VP1 and VP2.

Example 2 Isolation of Full Length 193P1E1B Encoding cDNA

To isolate genes that are involved in prostate cancer, an experiment wasconducted using the prostate cancer xenograft LAPC-9AD². The gene193P1E1B was derived from a subtraction consisting of a prostate cancerxenograft LAPC-9AD² minus prostate cancer xenograft LAPC-9AD. The SSHDNA sequence (FIG. 1) was designated 193P1E1B. Thirteen variants of193P1E1l were identified (FIGS. 2 and 3). cDNA clone 193P1E1B v.1 and193P1E1B v.5 were cloned from bladder cancer pool cDNA. 193P1E1B v.9 wascloned from LAPC-4AD cDNA library. All other variants were identified bybioinformatic analysis.

193P1E1B v.1 through v.8 differ from each other by one nucleic acidsubstitution. 193P1E1B v.1, v.2, v.4 v.7 and v.8 code for the sameprotein, whereas 193P1E1B v.5 and v.6 contain one amino acidsubstitution as shown in FIG. 12.

Absence of a 62-nucleotide sequence was identified in 193P1E1B v.9,nucleic acid positions 907-969 of 193P1E1B v.1. This resulted in an82-amino acid truncation at the amino terminus of 193P1E1B v.9. Othersplice variants were identified and referred to as 193P1E1B v.10, v.11,v.12 and v.13. 193P1E1B v.1, v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.11,v.12 and v.13 are novel proteins and have not been previously described.193P1E1B v.9 shows 99% identity to a hypothetical protein, MGC4832.193P1E1B v.10 shows 100% identity to a novel unnamed protein BAC03484.1.

Example 3 Chromosomal Mapping of 193P1E1B

Chromosomal localization can implicate genes in disease pathogenesis.Several chromosome mapping approaches are available includingfluorescent in situ hybridization (FISH), human/hamster radiation hybrid(RH) panels (Walter et al., 1994; Nature Genetics 7:22; ResearchGenetics, Huntsville Ala.), human-rodent somatic cell hybrid panels suchas is available from the Coriell Institute (Camden, N.J.), and genomicviewers utilizing BLAST homologies to sequenced and mapped genomicclones (NCBI, Bethesda, Md.).

193P1E1B maps to chromosome 13q11, using 193P1E1B sequence and the NCBIBLAST tool. This 13q11 region has been previously implicated in bladdercancer (Wada T, Louhelainen J, Hemminki K, Adolfsson J, Wijkstrom H,Norming U, Borgstrom E, Hansson J, Sandstedt B, Steineck G. Bladdercancer: allelic deletions at and around the retinoblastoma tumorsuppressor gene in relation to stage and grade. Clin Cancer Res. 2000Feb;6(2):610-5.).

Example 4 Expression Analysis of 193P1E1B

Expression of 193P1E1B was analyzed using 2 sets of primers asillustrated in FIG. 14A. First strand cDNA was prepared from vital pool1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon andstomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD,LAPC-9AI), normal thymus, prostate cancer pool, bladder cancer pool,kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancerpool, breast cancer pool, metastasis cancer pool, pancreas cancer pool,and from prostate cancer metastasis to lymph node from 2 differentpatients. Normalization was performed by PCR using primers to actin andGAPDH. Semi-quantitative PCR, using Primer Set A (B) or Primer Set B (C)to 193P1E1B, was performed at 30 cycles of amplification. A schematicdiagram depicting the location of the 2 primer sets A and B is shown inFIG. 14A. Primer Set A detected a PCR product of 190 bp which isidentical in all variants of 193P1E1B (FIG. 14B). Expression of 193P1E1Bwas observed in prostate cancer xenograft pool, prostate cancer pool,bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancerpool, ovary cancer pool, breast cancer pool, metastasis cancer pool,pancreas cancer pool, as well as the 2 prostate metastasis to lymphnode, but not in VP1 and VP2 (FIG. 14B). In order to test abundance ofexpression of 193P1E1B v.1 through v.8 compared to 193P1E1B v.9, anexperiment was conducted in which RT-PCR was performed using Primer SetB (FIG. 14C). Primer Set B detected a PCR product of 239 bp from193P1E1B v.1 through v.8, and of 177 bp from 193P1E1B v.9 (FIG. 14C).FIG. 14C shows that the transcipt encoding encoding 193P1E1B v.1 throughv.8, is expressed ate higher levels that the transcript encoding193P1E1B v.9. But both transcripts are expressed at similar proportionin all tissues tested.

Extensive northern blot analysis of 193P1E1B in 16 human normal tissuesconfirms the expression observed by RT-PCR (FIG. 15). Two transcripts ofapproximately 3.5 kb and 2 kb are only detected in testis and thymus,but not in any other normal tissue tested.

FIG. 16 shows expression of 193P1E1B in prostate cancer xenografts. RNAwas extracted from normal prostate, and from prostate cancer xenografts,LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 ug oftotal RNA/lane was probed with 193P1E1B SSH sequence. Northern blotanalysis shows expression of 193P1E1B in all 4 tissues, LAPC4AD,LAPC-4AI, LAPC-9AD, LAPC-9AI, with the lowest expression detected in theLAPC-9AD tissue, but not in normal prostate.

To test expression of 193P1E1B in patient cancer specimens, RNA wasextracted from a pool of three patients for each of the following,bladder cancer, colon cancer, ovary cancer and metastasis cancer, aswell as from normal prostate (NP), normal bladder (NB), normal kidney(NK), normal colon (NC). Northern blots with 10 ug of total RNA/lanewere probed with 193P1E1B SSH sequence (FIG. 17). Results showexpression of 193P1E1B in bladder cancer pool, colon cancer pool, ovarycancer pool and metastasis cancer pool, but not in any of the normaltissues tested.

Analysis of individual bladder cancer tissues by northern blot showsexpression of 193P1E1B in the 2 bladder cancer cell lines and in the 3bladder cancer patient specimens tested, but not in normal bladdertissues (FIG. 18).

FIG. 19 shows expression of 193P1E1Bin cancer metastasis patientspecimens. RNA was extracted from the following cancer metastasistissues, colon metastasis to lung, lung metastasis to lymph node, lungmetastasis to skin, and breast metastasis to lymph node, as well as fromnormal bladder (NB), normal lung (NL), normal breast (NBr), and normalovary (NO). Northern blots with 10 ug of total RNA/lane were probed with193P1E1B sequence. Size standards in kilobases (kb) are indicated on theside. The results show expression of 193P1E1B in all four differentcancer metastasis samples but not in the normal tissues tested.

FIG. 20 shows expression of 193P1E1B in pancreatic, ovarian andtesticular cancer patient specimens. RNA was extracted from pancreaticcancer (P1), ovarian cancer (P2, P3), and testicular cancer (P4, P5)isolated from cancer patients, as well as from normal pancreas(NPa).Northern blots with 10 ug of total RNA/lane were probed with 193P1E1Bsequence. Size standards in kilobases (kb) are indicated on the side.The results show expression of 193P1E1B in pancreatic, ovarian andtesticular cancer specimens but not in normal pancreas.

FIG. 21 shows expression of 193P1E1B in normal compared to patientcancer specimens. First strand cDNA was prepared from a panel of normaltissues (stomach, brain, heart, liver, spleen, skeletal muscle, testisprostate, bladder, kidney, colon, lung and pancreas) and from a panel ofpatient cancer pools (prostate cancer pool, bladder cancer pool, kidneycancer pool, colon cancer pool, lung cancer pool, pancreas cancer pool,ovary cancer pool, breast cancer pool, metastasis cancer pool, LAPCprostate xenograft pool (XP), and from prostate cancer metastasis tolymph node from 2 different patients (PMLN2). Normalization wasperformed by PCR using primers to actin. Semi-quantitative PCR, usingprimer Set A as described in FIG. 14, was performed was performed at 26and 30 cycles of amplification. Samples were run on an agarose gel, andPCR products were quantitated using the Alphalmager software. Relativeexpression was calculated by normalizing to signal obtained using actinprimers. Results show restricted 193P1E1B expression in normal testisamongst all normal tissues tested. 193P1E1B expression was stronglyupregulated in cancers of the bladder, colon, lung, pancreas, ovary,breast, and to a lesser extent in prostate and kidney cancers.

193P1E1B was also shown to be expressed in uterus, melanoma and bonecancer patient specimens. First strand cDNA was prepared from a panel ofuterus patient cancer specimens (A), melanoma and bone cancer specimens(B). Semi-quantitative PCR, using primers to 193P1E1B, was performed at26 and 30 cycles of amplification. Samples were run on an agarose gel,and PCR products were quantitated using the Alphalmager software.Expression was recorded as absent, low, or strong. Results showexpression of 193P1E1B in the majority of uterus patient cancerspecimens tested, as well as in the 2 melanoma specimens and in the bonetumor tested.

193P1E1B expression is reminiscent of a cancer-testis gene. Itsrestricted normal tissue expression to normal testis, and theupregulation detected in prostate cancer, bladder cancer, kidney cancer,colon cancer, lung cancer, ovary cancer, breast cancer and pancreaticcancer suggest that 193P1E1B is a potential therapeutic target and adiagnostic marker for human cancers.

Example 5 Transcript Variants of 193P1E1B

Transcript variants are variants of mature mRNA from the same gene whicharise by alternative transcription or alternative splicing. Alternativetranscripts are transcripts from the same gene but start transcriptionat different points. Splice variants are mRNA variants spliceddifferently from the same transcript. In eukaryotes, when a multi-exongene is transcribed from genomic DNA, the initial RNA is spliced toproduce functional mRNA, which has only exons and is used fortranslation into an amino acid sequence. Accordingly, a given gene canhave zero to many alternative transcripts and each transcript can havezero to many splice variants. Each transcript variant has a unique exonmakeup, and can have different coding and/or non-coding (5′ or 3′ end)portions, from the original transcript. Transcript variants can code forsimilar or different proteins with the same or a similar function or canencode proteins with different functions, and can be expressed in thesame tissue at the same time, or in different tissues at the same time,or in the same tissue at different times, or in different tissues atdifferent times. Proteins encoded by transcript variants can havesimilar or different cellular or extracellular localizations, e.g.,secreted versus intracellular.

Transcript variants are identified by a variety of art-accepted methods.For example, alternative transcripts and splice variants are identifiedby full-length cloning experiment, or by use of full-length transcriptand EST sequences. First, all human ESTs were grouped into clusterswhich show direct or indirect identity with each other. Second, ESTs inthe same cluster were further grouped into sub-clusters and assembledinto a consensus sequence. The original gene sequence is compared to theconsensus sequence(s) or other full-length sequences. Each consensussequence is a potential splice variant for that gene. Even when avariant is identified that is not a full-length clone, that portion ofthe variant is very useful for antigen generation and for furthercloning of the full-length splice variant, using techniques known in theart.

Moreover, computer programs are available in the art that identifytranscript variants based on genomic sequences. Genomic-based transcriptvariant identification programs include FgenesH (A. Salamov and V.Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” GenomeResearch. April 2000; 10(4):516-22); Grail and GenScan. For a generaldiscussion of splice variant identification protocols see., e.g.,Southan, C., A genomic perspective on human proteases, FEBS Lett. Jun.8, 2001; 498(2-3):214-8; de Souza, S. J., et al., Identification ofhuman chromosome 22 transcribed sequences with ORF expressed sequencetags, Proc. Natl. Acad. Sci USA. Nov. 7, 2000; 97(23):12690-3.

To further confirm the parameters of a transcript variant, a variety oftechniques are available in the art, such as full-length cloning,proteomic validation, PCR-based validation, and 5′ RACE validation, etc.(see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin bankspeninsula: a new termination variant characterized by electrospray massspectrometry, Biochem Biophys Acta. Aug. 17, 1999;1433(1-2):321-6;Ferranti P, et al., Differential splicing of pre-messenger RNA producesmultiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. Oct.1, 1997;249(1):1-7. For PCR-based Validation: Wellmann S, et al.,Specific reverse transcription-PCR quantification of vascularendothelial growth factor (VEGF) splice variants by LightCyclertechnology, Clin Chem. April 2001;47(4):654-60; Jia, H. P., et al.,Discovery of new human beta-defensins using a genomics-based approach,Gene. Jan. 24, 2001; 263(1-2):211-8. For PCR-based and 5′ RACEValidation: Brigle, K. E., et al., Organization of the murine reducedfolate carrier gene and identification of variant splice forms, BiochemBiophys Acta. Aug. 7, 1997; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancers.When the genomic region to which a gene maps is modulated in aparticular cancer, the alternative transcripts or splice variants of thegene are modulated as well. Disclosed herein is that 193P1E1B has aparticular expression profile related to cancer. Alternative transcriptsand splice variants of 193P1E1B may also be involved in cancers in thesame or different tissues, thus serving as tumor-associatedmarkers/antigens.

Using the full-length gene and EST sequences, three transcript variantswere identified, designated as 193P1E1B v.7, v.8 and v.9. Compared with193P1E1B v.1, transcript variant 193P1E1B v.7 has spliced out exons 10and 11 from variant 193P1E1B v.1, as shown in FIG. 12. Variant 193P1E1Bv.8 inserted 36 bp in between 1931 and 1932 of variant 193P1E1B v.1 andvariant 193P1E1B v.9 replaced with 36 bp the segment 1136-1163 ofvariant 193P1E1B v.1. Theoretically, each different combination of exonsin spatial order, e.g. exons 2 and 3, is a potential splice variant.Tables LI through LXX are set forth on a variant-by-variant bases.Tables LI, LV, LIX, LXIII, and LXVII show the nucleotide sequence of thetranscript variant. Tables LII, LVI, LX, LXIV, and LXVIII show thealignment of the transcript variant with nucleic acid sequence of193P1E1B v.1. Tables LIII, LVII, LXI, LXV, and LXIX show the amino acidtranslation of the transcript variant for the identified reading frameorientation. Tables LIV, LVIII, LXII, LXVI, and LXX display alignmentsof the amino acid sequence encoded by the splice variant with that of193P1E1Bv.1.

Example 6 Single Nucleotide Polymorphisms of 193P1E1B

A Single Nucleotide Polymorphism (SNP) is a single base pair variationin a nucleotide sequence at a specific location. At any given point ofthe genome, there are four possible nucleotide base pairs: A/T, CIG, G/Cand T/A. Genotype refers to the specific base pair sequence of one ormore locations in the genome of an individual. Haplotype refers to thebase pair sequence of more than one location on the same DNA molecule(or the same chromosome in higher organisms), often in the context ofone gene or in the context of several tightly linked genes. SNPs thatoccur on a cDNA are called cSNPs. These cSNPs may change amino acids ofthe protein encoded by the gene and thus change the functions of theprotein. Some SNPs cause inherited diseases; others contribute toquantitative variations in phenotype and reactions to environmentalfactors including diet and drugs among individuals. Therefore, SNPsand/or combinations of alleles (called haplotypes) have manyapplications, including diagnosis of inherited diseases, determinationof drug reactions and dosage, identification of genes responsible fordiseases, and analysis of the genetic relationship between individuals(P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect humantraits,” Curr. Opin. Neurobiol. October 2001; 11(5):637-641; M.Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drugreactions,” Trends Pharmacol. Sci. June 2001; 22(6):298-305; J. H.Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotidepolymorphisms in the isolation of common disease genes,”Pharmacogenomics. February 2000; 1(1):3947; R. Judson, J. C. Stephensand A. Windemuth, “The predictive power of haplotypes in clinicalresponse,” Pharmacogenomics. 2000 feb; 1(1):15-26).

SNPs are identified by a variety of art-accepted methods (P. Bean, “Thepromising voyage of SNP target discovery,” Am. Clin. Lab.October-November 2001; 20(9):18-20; K. M. Weiss, “In search of humanvariation,” Genome Res. July 1998; 8(7):691-697; M. M. She, “Enablinglarge-scale pharmacogenetic studies by high-throughput mutationdetection and genotyping technologies,” Clin. Chem. 2001 Feb;47(2):164-172). For example, SNPs are identified by sequencing DNAfragments that show polymorphism by gel-based methods such asrestriction fragment length polymorphism (RFLP) and denaturing gradientgel electrophoresis (DGGE). They can also be discovered by directsequencing of DNA samples pooled from different individuals or bycomparing sequences from different DNA samples. With the rapidaccumulation of sequence data in public and private databases, one candiscover SNPs by comparing sequences using computer programs (Z. Gu, L.Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting incyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified andgenotype or haplotype of an individual can be determined by a variety ofmethods including direct sequencing and high throughput microarrays (P.Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu.Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K.Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft,“High-throughput SNP genotyping with the Masscode system,” Mol. Diagn.December 2000; 5(4):329-340). Using the methods described above, sevenSNPs were identified in the original transcript, 193P1E1B v.1, atpositions 57 (A/G), 792 (C/G), 804 (C/A), 1253 (G/A), 1564 (A/G), 2268(C/T) and 2387 (C/T). The transcripts or proteins with alternativealleles were designated as variants 193P1E1B v.2, v.3, v.4, v.5 and v.6,respectively. FIG. 10 shows the schematic alignment of the SNP variants.FIG. 11 shows the schematic alignment of protein variants, correspondingto nucleotide variants. Nucleotide variants that code for the same aminoacid sequence as variant 1 are not shown in FIG. 11. These alleles ofthe SNPs, though shown separately here, can occur in differentcombinations (haplotypes) and in any one of the transcript variants(such as 193P1E1B v.9) that contains the sequence context of the SNPs.

Example 7 Production of Recombinant 193P1E1B in Prokaryotic Systems

To express recombinant 193P1E1B in prokaryotic cells, the full orpartial length 193P1E1B cDNA sequences can be cloned into any one of avariety of expression vectors known in the art. One or more of thefollowing regions of 193P1E1B are expressed in these constructs, aminoacids 1 to 412 of variant 5 or variant 2; or amino acids 1 to 388 ofvariant 10, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from193P1E1B, variants, or analogs thereof. In certain embodiments a regionof 193P1E1B is expressed that encodes an amino acid not shared amongstat least variants.

A. In vitro transcription and translation constructs:

pCRII: To generate 193P1E1B sense and anti-sense RNA probes for RNA insitu investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) aregenerated encoding either all or fragments of a 193P1E1B cDNA. The pCRIIvector has Sp6 and T7 promoters flanking the insert to drive thetranscription of 193P1E1B RNA for use as probes in RNA in situhybridization experiments. These probes are used to analyze the cell andtissue expression of 193P1E1B at the RNA level. Transcribed 193P1E1B RNArepresenting the cDNA amino acid coding region of the 193P1E1B gene isused in in vitro translation systems such as the TnT™ CoupledReticulolysate System (Promega, Corp., Madison, Wis.) to synthesize193P1E1B protein.

B. Bacterial Constructs:

PGEX Constructs: To generate recombinant 193P1E1B proteins in bacteriathat are fused to the Glutathione S-transferase (GST) protein, all orparts of a 193P1E1B cDNA protein coding sequence are fused to the GSTgene by cloning into pGEX-6P-1 or any other GST-fusion vector of thepGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). Theseconstructs allow controlled expression of recombinant 193P1E1B proteinsequences with GST fused at the amino-terminus and a six histidineepitope (6×His) at the carboxyl-terminus. The GST and 6×His tags permitpurification of the recombinant fusion protein from induced bacteriawith the appropriate affinity matrix and allow recognition of the fusionprotein with anti-GST and anti-His antibodies. The 6×His tag isgenerated by adding 6 histidine codons to the cloning primer at the 3′end, e.g., of the open reading frame (ORF). A proteolytic cleavage site,such as the PreScission™ recognition site in pGEX-6P-1, may be employedsuch that it permits cleavage of the GST tag from 193P1E1B-relatedprotein. The ampicillin resistance gene and pBR322 origin permitsselection and maintenance of the pGEX plasmids in E. coli.

pMAL Constructs: To generate, in bacteria, recombinant 193P1E1B proteinsthat are fused to maltose-binding protein (MBP), all or parts of a193P1E1B cDNA protein coding sequence are fused to the MBP gene bycloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs,Beverly, Mass.). These constructs allow controlled expression ofrecombinant 193P1E1B protein sequences with MBP fused at theamino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBPand 6×His tags permit purification of the recombinant protein frominduced bacteria with the appropriate affinity matrix and allowrecognition of the fusion protein with anti-MBP and anti-His antibodies.The 6×His epitope tag is generated by adding 6 histidine codons to the3′ cloning primer. A Factor Xa recognition site permits cleavage of thepMAL tag from 193P1E1B. The pMAL-c2X and pMAL-p2X vectors are optimizedto express the recombinant protein in the cytoplasm or periplasmrespectively. Periplasm expression enhances folding of proteins withdisulfide bonds.

pET Constructs: To express 193P1E1B in bacterial cells, all or parts ofa 193P1E1B cDNA protein coding sequence are cloned into the pET familyof vectors (Novagen, Madison, Wis.). These vectors allow tightlycontrolled expression of recombinant 193P1E1B protein in bacteria withand without fusion to proteins that enhance solubility, such as NusA andthioredoxin (Trx), and epitope tags, such as 6×His and S-Tag Tm that aidpurification and detection of the recombinant protein. For example,constructs are made utilizing pET NusA fusion system 43.1 such thatregions of a 193P1E1B protein are expressed as amino-terminal fusions toNusA.

C. Yeast Constructs:

pESC Constructs: To express 193P1E1B in the yeast species Saccharomycescerevisiae for generation of recombinant protein and functional studies,all or parts of a 193P1E1B cDNA protein coding sequence are cloned intothe pESC family of vectors each of which contain 1 of 4 selectablemarkers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.).These vectors allow controlled expression from the same plasmid of up to2 different genes or cloned sequences containing either Flag™ or Mycepitope tags in the same yeast cell. This system is useful to confirmprotein-protein interactions of 193P1E1B. In addition, expression inyeast yields similar post-translational modifications, such asglycosylations and phosphorylations, that are found when expressed ineukaryotic cells.

pESP Constructs: To express 193P1E1B in the yeast species Saccharomycespombe, all or parts of a 193P1E1B cDNA protein coding sequence arecloned into the pESP family of vectors. These vectors allow controlledhigh level of expression of a 193P1E1B protein sequence that is fused ateither the amino terminus or at the carboxyl terminus to GST which aidspurification of the recombinant protein. A Flag™ epitope tag allowsdetection of the recombinant protein with anti-Flag™ antibody.

Example 8 Production of Recombinant 193P1E1B in Higher EukarvoticSystems

A. Mammalian Constructs:

To express recombinant 193P1E1B in eukaryotic cells, the full or partiallength 193P1E1B cDNA sequences can be cloned into any one of a varietyof expression vectors known in the art. One or more of the followingregions of 193P1E1B are expressed in these constructs, amino acids 1 to412; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from193P1E1B, variants, or analogs thereof.

The constructs can be transfected into any one of a wide variety ofmammalian cells such as 293T cells. Transfected 293T cell lysates can beprobed with the anti-193P1E1B polyclonal serum, described herein.

pcDNA4/HisMax Constructs: To express 193P1E1B in mammalian cells, the193P1E1B ORF, or portions thereof, of 193P1E1B are cloned intopcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Proteinexpression is driven from the cytomegalovirus (CMV) promoter and theSP16 translational enhancer. The recombinant protein has Xpress™ and sixhistidine (6×His) epitopes fused to the amino-terminus. ThepcDNA4/HisMax vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheZeocin resistance gene allows for selection of mammalian cellsexpressing the protein and the ampicillin resistance gene and ColE1origin permits selection and maintenance of the plasmid in E. coli.

pcDNA3.11MycHis Constructs: To express 193P1E1B in mammalian cells, the193P1E1B ORF, or portions thereof, of 193P1E1B with a consensus Kozaktranslation initiation site are cloned into pcDNA3.1/MycHis Version A(Invitrogen, Carlsbad, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the mycepitope and 6×His epitope fused to the carboxyl-terminus. ThepcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability, along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene can be used, as it allows for selection ofmammalian cells expressing the protein and the ampicillin resistancegene and ColE1 origin permits selection and maintenance of the plasmidin E. coli.

pcDNA3.1/CT-GFP-TOPO Construct: To express 193P1E1B in mammalian cellsand to allow detection of the recombinant proteins using fluorescence,the 193P1E1B ORF, or portions thereof, of 193P1E1B with a consensusKozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO(Invitrogen, Calif.). Protein expression is driven from thecytomegalovirus (CMV) promoter. The recombinant proteins have the GreenFluorescent Protein (GFP) fused to the carboxyl-terminus facilitatingnon-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH)polyadenylation signal and transcription termination sequence to enhancemRNA stability along with the SV40 origin for episomal replication andsimple vector rescue in cell lines expressing the large T antigen. TheNeomycin resistance gene allows for selection of mammalian cells thatexpress the protein, and the ampicillin resistance gene and ColE1 originpermits selection and maintenance of the plasmid in E. coli. Additionalconstructs with an amino-terminal GFP fusion are made inpcDNA3.1/NT-GFP-TOPO spanning the entire length of the 193P1E1Bproteins.

PAPtag: The 193P1E1B ORF, or portions thereof, of 193P1E1B are clonedinto pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This constructgenerates an alkaline phosphatase fusion at the carboxyl-terminus of the193P1E1B proteins while fusing the IgGκ signal sequence to theamino-terminus. Constructs are also generated in which alkalinephosphatase with an amino-terminal IgGκ signal sequence is fused to theamino-terminus of 193P1E1B proteins. The resulting recombinant 193P1E1Bproteins are optimized for secretion into the media of transfectedmammalian cells and can be used to identify proteins such as ligands orreceptors that interact with the 193P1E1B proteins. Protein expressionis driven from the CMV promoter and the recombinant proteins alsocontain myc and 6×His epitopes fused at the carboxyl-terminus thatfacilitates detection and purification. The Zeocin resistance genepresent in the vector allows for selection of mammalian cells expressingthe recombinant protein and the ampicillin resistance gene permitsselection of the plasmid in E. coli.

ptag5: The 193P1E1B ORF, or portions thereof, of 193P1E1B are clonedinto pTag-5. This vector is similar to pAPtag but without the alkalinephosphatase fusion. This construct generates 193P1E1B protein with anamino-terminal IgGκ signal sequence and myc and 6×His epitope tags atthe carboxyl-terminus that facilitate detection and affinitypurification. The resulting recombinant 193P1E1B protein is optimizedfor secretion into the media of transfected mammalian cells, and is usedas immunogen or ligand to identify proteins such as ligands or receptorsthat interact with the 193P1E1B proteins. Protein expression is drivenfrom the CMV promoter. The Zeocin resistance gene present in the vectorallows for selection of mammalian cells expressing the protein, and theampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc: The 193P1E1B ORF, or portions thereof, of 193P1E1B are alsocloned into psecFc. The psecFc vector was assembled by cloning the humanimmunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2(Invitrogen, California). This construct generates an IgG1 Fc fusion atthe carboxyl-terminus of the 193P1E1B proteins, while fusing the IgGKsignal sequence to N-terminus. 193P1E1B fusions utilizing the murineIgG1 Fc region are also used. The resulting recombinant 193P1E1Bproteins are optimized for secretion into the media of transfectedmammalian cells, and can be used as immunogens or to identify proteinssuch as ligands or receptors that interact with the 193P1E1B protein.Protein expression is driven from the CMV promoter. The hygromycinresistance gene present in the vector allows for selection of mammaliancells that express the recombinant protein, and the ampicillinresistance gene permits selection of the plasmid in E. coli.

pSRα Constructs: To generate mammalian cell lines that express 193P1E1Bconstitutively, 193P1E1B ORF, or portions thereof, of 193P1E1B arecloned into pSRα constructs. Amphotropic and ecotropic retroviruses aregenerated by transfection of pSRα constructs into the 293T-10A1packaging line or co-transfection of pSRα and a helper plasmid(containing deleted packaging sequences) into the 293 cells,respectively. The retrovirus is used to infect a variety of mammaliancell lines, resulting in the integration of the cloned gene, 193P1E1B,into the host cell-lines. Protein expression is driven from a longterminal repeat (LTR). The Neomycin resistance gene present in thevector allows for selection of mammalian cells that express the protein,and the ampicillin resistance gene and ColE1 origin permit selection andmaintenance of the plasmid in E. coli. The retroviral vectors canthereafter be used for infection and generation of various cell linesusing, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Additional pSRα constructs are made that fuse an epitope tag such as theFLAG™ tag to the carboxyl-terminus of 193P1E1B sequences to allowdetection using anti-Flag antibodies. For example, the FLAG™ sequence 5′gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 62) is added to cloningprimer at the 3′ end of the ORF. Additional pSRα constructs are made toproduce both amino-terminal and carboxyl-terminal GFP and myc/6×Hisfusion proteins of the full-length 193P1E1B proteins.

Additional Viral Vectors: Additional constructs are made forviral-mediated delivery and expression of 193P1E1B. High virus titerleading to high level expression of 193P1E1B is achieved in viraldelivery systems such as adenoviral vectors and herpes amplicon vectors.The 193P1E1B coding sequences or fragments thereof are amplified by PCRand subcloned into the AdEasy shuffle vector (Stratagene). Recombinationand virus packaging are performed according to the manufacturer'sinstructions to generate adenoviral vectors. Alternatively, 193P1E1Bcoding sequences or fragments thereof are cloned into the HSV-1 vector(Imgenex) to generate herpes viral vectors. The viral vectors arethereafter used for infection of various cell lines such as PC3, NIH3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control expression of 193P1E1B inmammalian cells, coding sequences of 193P1E1B, or portions thereof, arecloned into regulated mammalian expression systems such as the T-RexSystem (Invitrogen), the GeneSwitch System (Invitrogen) and thetightly-regulated Ecdysone System (Sratagene). These systems allow thestudy of the temporal and concentration dependent effects of recombinant193P1E1B. These vectors are thereafter used to control expression of193P1E1B in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems

To generate recombinant 193P1E1B proteins in a baculovirus expressionsystem, 193P1E1B ORF, or portions thereof, are cloned into thebaculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides aHis-tag at the N-terminus. Specifically, pBlueBac-193P1E1B isco-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9(Spodoptera frugiperda) insect cells to generate recombinant baculovirus(see Invitrogen instruction manual for details). Baculovirus is thencollected from cell supernatant and purified by plaque assay.

Recombinant 193P1E1B protein is then generated by infection of HighFiveinsect cells (Invitrogen) with purified baculovirus. Recombinant193P1E1B protein can be detected using anti-193P1E1B or anti-His-tagantibody. 193P1E1B protein can be purified and used in variouscell-based assays or as immunogen to generate polyclonal and monoclonalantibodies specific for 193P1E1B.

Example 9 Antigenicity Profiles and Secondary Structure

FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five aminoacid profiles of the 193P1E1B amino acid sequence (variant 1), eachassessment is available by accessing the ProtScale website on the ExPasymolecular biology server.

These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981.Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity,(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7,Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG.8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int.J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., RouxB. 1987 Protein Engineering 1:289-294); and optionally others availablein the art, such as on the ProtScale website, were used to identifyantigenic regions of 193P1E1B protein. Each of the above amino acidprofiles of 193P1E1B were generated using the following ProtScaleparameters for analysis: 1) A window size of 9; 2) 100% weight of thewindow edges compared to the window center; and, 3) amino acid profilevalues normalized to lie between 0 and 1.

Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and PercentageAccessible Residues (FIG. 7) profiles were used to determine stretchesof hydrophilic amino acids (i.e., values greater than 0.5 on theHydrophilicity and Percentage Accessible Residues profiles, and valuesless than 0.5 on the Hydropathicity profile). Such regions are likely tobe exposed to the aqueous environment, be present on the surface of theprotein, and thus available for immune recognition, such as byantibodies.

Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determinestretches of amino acids (i.e., values greater than 0.5 on the Beta-turnprofile and the Average Flexibility profile) that are not constrained insecondary structures such as beta sheets and alpha helices. Such regionsare also more likely to be exposed on the protein and thus accessible toimmune recognition, such as by antibodies.

Antigenic sequences of the full length 193P1E1B protein (variant 1)indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7,FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides ornucleic acids that encode them, to generate therapeutic and diagnosticanti-193P1E1B antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50 or more than 50 contiguous amino acids, or the correspondingnucleic acids that encode them, from 193P1E1B protein. In particular,peptide immunogens of the invention can comprise, a peptide region of atleast 5 amino acids of FIG. 2 in any whole number increment up to 412that includes an amino acid position having a value greater than 0.5 inthe Hydrophilicity profile of FIG. 5; a peptide region of at least 5amino acids of FIG. 2 in any whole number increment up to 412 thatincludes an amino acid position having a value less than 0.5 in theHydropathicity profile of FIG. 6; a peptide region of at least 5 aminoacids of FIG. 2 in any whole number increment up to 412 that includes anamino acid position having a value greater than 0.5 in the PercentAccessible Residues profile of FIG. 7; a peptide region of at least 5amino acids of FIG. 2 in any whole number increment up to 412 thatincludes an amino acid position having a value greater than 0.5 in theAverage Flexibility profile on FIG. 8; and, a peptide region of at least5 amino acids of FIG. 2 in any whole number increment up to 412 thatincludes an amino acid position having a value greater than 0.5 in theBeta-turn profile of FIG. 9. Peptide immunogens of the invention canalso comprise nucleic acids that encode any of the forgoing.

All immunogens of the invention, peptide or nucleic acid, can beembodied in human unit dose form, or comprised by a composition thatincludes a pharmaceutical excipient compatible with human physiology.

The secondary structure of 193P1E1B, namely the predicted presence andlocation of alpha helices, extended strands, and random coils, ispredicted from the primary amino acid sequence of 193P1E1B variant 1using the HNN-Hierarchical Neural Network method, accessed from theExPasy molecular biology server. The analysis indicates that 193P1E1B iscomposed 29.13% alpha helix, 9.95% extended strand, and 60.92% randomcoil (FIG. 13).

Analysis of 193P1E1B using a variety of transmembrane predictionalgorithms accessed from the ExPasy molecular biology server did notpredict the presence of such domains, confirming that 193P1E1B is asoluble protein.

Example 10 Generation of 193P1E1B Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one ormore injections of an immunizing agent and, if desired, an adjuvant.Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections. Inaddition to immunizing with the full length 193P1E1B protein, computeralgorithms are employed in design of immunogens that, based on aminoacid sequence analysis contain characteristics of being antigenic andavailable for recognition by the immune system of the immunized host(see Example 9 entitled “Antigenicity Profiles and SecondaryStructure”). Such regions would be predicted to be hydrophilic,flexible, in beta-turn conformations, and be exposed on the surface ofthe protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 foramino acid profiles that indicate such regions of 193P1E1B).

For example, 193P1E1B recombinant bacterial fusion proteins or peptidescontaining hydrophilic, flexible, beta-turn regions of 193P1E1B are usedas antigens to generate polyclonal antibodies in New Zealand Whiterabbits. For example, such regions include, but are not limited to,amino acids 20-43, amino acids 100-164, amino acids 241-261, or aminoacids 310-331. It is useful to conjugate the immunizing agent to aprotein known to be immunogenic in the mammal being immunized. Examplesof such immunogenic proteins include, but are not limited to, keyholelimpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, andsoybean trypsin inhibitor. In one embodiment, a peptide encoding aminoacids 241-261 of 193P1E1B is conjugated to KLH and used to immunize therabbit. Alternatively the immunizing agent may include all or portionsof a 193P1E1B protein, analogs or fusion proteins thereof. For example,a 193P1E1B amino acid sequence can be fused using recombinant DNAtechniques to any one of a variety of fusion protein partners that arewell known in the art, such as glutathione-S-transferase (GST) and HIStagged fusion proteins. Such fusion proteins are purified from inducedbacteria using the appropriate affinity matrix.

In one embodiment, a GST-fusion protein containing an entire 193P1E1Bcoding sequence is produced and purified and used as immunogen. Otherrecombinant bacterial fusion proteins that can be employed includemaltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulinconstant region (see the section entitled “Production of 193P1E1B inProkaryotic Systems” and Current Protocols In Molecular Biology, Volume2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S.,Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L.(1991)J.Exp. Med. 174, 561-566).

In addition to bacterial-derived fusion proteins, mammalian-expressedprotein antigens are also used. These antigens are expressed frommammalian expression vectors such as the Tag5 and Fc-fusion vectors (seeExample 8 entitled “Production of Recombinant 193P1E1B in EukaryoticSystems”), and retain post-translational modifications such asglycosylations found in native protein. In one embodiment, an entire193P1E1B coding sequence is cloned into the Tag5 mammalian secretionvector. The recombinant protein is purified by metal chelatechromatography from tissue culture supernatants of 293T cells stablyexpressing the recombinant vector. The purified Tag5 193P1E1B protein isthen used as immunogen.

During the immunization protocol, it is useful to mix or emulsify theantigen in adjuvants that enhance the immune response of the hostanimal. Examples of adjuvants include, but are not limited to, completeFreund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate).

In a typical protocol, rabbits are initially immunized subcutaneouslywith up to 200 μg, typically 100-200 μg, of fusion protein or peptideconjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits arethen injected subcutaneously every two weeks with up to 200 μg,typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant(IFA). Test bleeds are taken approximately 7-10 days following eachimmunization and used to monitor the titer of the antiserum by ELISA.

To test reactivity and specificity of immune serum, such as the rabbitserum derived from immunization with Tag5 193P1E1B protein orKLH-coupled peptide encoding amino acids 241-261, the full-length193P1E1B cDNA is cloned into pCDNA 3.1 myc-his expression vector(invitrogen, see the Example entitled “Production of Recombinant193P1E1B in Eukaryotic Systems”). After transfection of the constructsinto 293T cells, cell lysates are probed with the anti-193P1E1B serumand with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz,Calif.) to determine specific reactivity to denatured 193P1E1B proteinusing the Western blot technique. Immunoprecipitation and flowcytometric analyses of 293T and other recombinant 193P1E1B-expressingcells determine recognition of native protein by the antiserum. Inaddition, Western blot, immunoprecipitation, fluorescent microscopy, andflow cytometric techniques using cells that endogenously express193P1E1B are carded out to test specificity.

The anti-serum from the Tag5 193P1E1B immunized rabbit is affinitypurified by passage over a column composed of the Tag5 antigencovalently coupled to Affigel matrix (BioRad, Hercules, Calif.). Theserum is then further purified by protein G affinity chromatography toisolate the IgG fraction. Serum from rabbits immunized with fusionproteins, such as GST and MBP fusion proteins, are purified by depletionof antibodies reactive to the fusion partner sequence by passage over anaffinity column containing the fusion partner either alone or in thecontext of an irrelevant fusion protein. Sera from other His-taggedantigens and peptide immunized rabbits as well as fusion partnerdepleted sera are affinity purified by passage over a column matrixcomposed of the original protein immunogen or free peptide.

Example 11 Generation of 193P1E1B Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to 193P1E1B comprise those thatreact with epitopes of the protein that would disrupt or modulate thebiological function of 193P1E1B, for example those that would disruptits interaction with ligands, proteins, or substrates that mediate itsbiological activity. Immunogens for generation of such mAbs includethose designed to encode or contain an entire 193P1E1B protein or itsvariants or regions of a193P1E1B protein predicted to be antigenic fromcomputer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6,FIG. 7, FIG. 8, or FIG. 9, and Example 9 entitled “Antigenicity Profilesand Secondary Structure”). Immunogens include peptides, recombinantbacterial proteins, and mammalian expressed Tag 5 proteins and human andmurine IgG FC fusion proteins. In addition, cells expressing high levelsof 193P1E1B, such as 293T-193P1E1B or 300.19-193P1E1B murine Pre-Bcells, are used to immunize mice.

To generate mAbs to 193P1E1B, mice are first immunized intraperitoneally(IP) with, typically, 10-50 μg of protein immunogen or 10⁷193P1E1B-expressing cells mixed in complete Freund's adjuvant. Mice arethen subsequently immunized IP every 2-4 weeks with, typically, 10-50 μgof protein immunogen or 10⁷ cells mixed in incomplete Freund's adjuvant.Alternatively, MPL-TDM adjuvant is used in immunizations. In addition tothe above protein and cell-based immunization strategies, a DNA-basedimmunization protocol is employed in which a mammalian expression vectorencoding 193P1E1B sequence is used to immunize mice by direct injectionof the plasmid DNA. For example, an entire coding sequence of 193P1E1B,e.g., amino acids 1-412 of 193P1E1B variant 1, is cloned into the Tag5mammalian secretion vector and the recombinant vector is used asimmunogen. In another example the amino acids are cloned into anFc-fusion secretion vector in which a 193P1E1B sequence is fused at theamino-terminus to an IgK leader sequence and at the carboxyl-terminus tothe coding sequence of the human or murine IgG Fc region. Thisrecombinant vector is then used as immunogen. The plasmid immunizationprotocols are used in combination with purified proteins expressed fromthe same vector and with cells expressing 193P1E1B.

During the immunization protocol, test bleeds are taken 7-10 daysfollowing an injection to monitor titer and specificity of the immuneresponse. Once appropriate reactivity and specificity is obtained asdetermined by ELISA, Western blotting, immunoprecipitation, fluorescencemicroscopy, and flow cytometric analyses, fusion and hybridomageneration is then carried out with established procedures well known inthe art (see, e.g., Harlow and Lane, 1988).

In one embodiment, monoclonal antibodies are derived that distinguishbetween, e.g., the various 193P1E1B variants, e.g., the amino terminaltruncated splice variant 3, encoding amino acids 83-412 and the fulllength protein encoding amino acids 1-412. In one method, two differentFc-fusion proteins are derived, one encoding amino acids 1-82, and theother encoding amino acids 83-412. These are expressed and purified fromstably transfected 293T cells. Balb C mice are initially immunizedintraperitoneally with 25 μg of the Tag5-193P1E1B protein mixed incomplete Freund's adjuvant. Mice are subsequently immunized every twoweeks with 25 μg of the antigen mixed in incomplete Freund's adjuvantfor a total of three immunizations. ELISA using the Tag5 antigendetermines the titer of serum from immunized mice. Reactivity andspecificity of serum to the full length 193P1E1B protein and to aminoterminal truncated variant 3 is monitored by Western blotting,immunoprecipitation and flow cytometry using 293T cells transfected withan expression vector encoding each of the respective 193P1E1B cDNAs (seee.g., the Example entitled “Production of Recombinant 193P1E1B inEukaryotic Systems”). Other recombinant 193P1E1B-expressing cells orcells endogenously expressing 193P1E1B are also used. Mice showing thestrongest reactivity are rested and given a final injection of Tag5antigen in PBS and then sacrificed four days later. The spleens of thesacrificed mice are harvested and fused to SPO/2 myeloma cells usingstandard procedures (see, e.g., Harlow and Lane, 1988). Supernatantsfrom HAT selected growth wells are screened by ELISA, Western blot,immunoprecipitation, fluorescent microscopy, and flow cytometry toidentify 193P1E1B specific antibody-producing clones.

The binding affinity of a 193P1E1B monoclonal antibody is determinedusing standard technologies. Affinity measurements quantify the strengthof antibody to epitope binding and are used to help define which193P1E1B monoclonal antibodies preferred, e.g., for diagnostic ortherapeutic use, as appreciated by one of skill in the art. The BIAcoresystem (Uppsala, Sweden) is a preferred method for determining bindingaffinity. The BIAcore system uses surface plasmon resonance (SPR,Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998,Methods in Enzymology 295: 268) to monitor biomolecular interactions inreal time. BIAcore analysis conveniently generates association rateconstants, dissociation rate constants, equilibrium dissociationconstants, and affinity constants.

Example 12 HLA Class I and Class II Binding Assays

HLA class I and class II binding assays using purified HLA molecules areperformed in accordance with disclosed protocols (e.g., PCT publicationsWO 94/20127 and WO 94/03205; Sidney et al., Current Protocols inImmunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995);Sette, et al., Mol. Immunol. 31:813 (1994)). Briefly, purified MHCmolecules (5 to 500 nM) are incubated with various unlabeled peptideinhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described.Following incubation, MHC-peptide complexes are separated from freepeptide by gel filtration and the fraction of peptide bound isdetermined. Typically, in preliminary experiments, each MHC preparationis titered in the presence of fixed amounts of radiolabeled peptides todetermine the concentration of HLA molecules necessary to bind 10-20% ofthe total radioactivity. All subsequent inhibition and direct bindingassays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≧[HLA], the measuredIC₅₀ values are reasonable approximations of the true KD values. Peptideinhibitors are typically tested at concentrations ranging from 120 μg/mlto 1.2 ng/ml, and are tested in two to four completely independentexperiments. To allow comparison of the data obtained in differentexperiments, a relative binding figure is calculated for each peptide bydividing the IC₅₀ of a positive control for inhibition by the IC₅₀ foreach tested peptide (typically unlabeled versions of the radiolabeledprobe peptide). For database purposes, and inter-experiment comparisons,relative binding values are compiled. These values can subsequently beconverted back into IC₅₀ nM values by dividing the IC₅₀ nM of thepositive controls for inhibition by the relative binding of the peptideof interest. This method of data compilation is accurate and consistentfor comparing peptides that have been tested on different days, or withdifferent lots of purified MHC.

Binding assays as outlined above may be used to analyze HLA supermotifand/or HLA motif-bearing peptides (see Table IV).

Example 13 Identification of HLA Supermotif-and Motif-Bearing CTLCandidate Epitopes

HLA vaccine compositions of the invention can include multiple epitopes.The multiple epitopes can comprise multiple HLA supermotifs or motifs toachieve broad population coverage. This example illustrates theidentification and confirmation of supermotif- and motif-bearingepitopes for the inclusion in such a vaccine composition. Calculation ofpopulation coverage is performed using the strategy described below.

Computer Searches and Algorithms for Identification of Supermotif and/orMotif-Bearing Epitopes

The searches performed to identify the motif-bearing peptide sequencesin the Example entitled “Antigenicity Profiles” and Tables VIII-XXI andXXII-XLIX employ the protein sequence data from the gene product of193P1E1B. set forth in FIGS. 2 and 3, the specific search peptides usedto generate the tables are listed in Table VII.

Computer searches for epitopes bearing HLA Class I or Class IIsupermotifs or motifs are performed as follows. All translated 193P1E1Bprotein sequences are analyzed using a text string search softwareprogram to identify potential peptide sequences containing appropriateHLA binding motifs; such programs are readily produced in accordancewith information in the art in view of known motif/supermotifdisclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored usingpolynomial algorithms to predict their capacity to bind to specificHLA-Class I or Class II molecules. These polynomial algorithms accountfor the impact of different amino acids at different positions, and areessentially based on the premise that the overall affinity (or AG) ofpeptide-HLA molecule interactions can be approximated as a linearpolynomial function of the type:“ΔG”=a _(1i) ×a _(2i) ×a _(3i) . . ×a _(ni)

where a_(ji) is a coefficient which represents the effect of thepresence of a given amino acid (j) at a given position (i) along thesequence of a peptide of n amino acids. The crucial assumption of thismethod is that the effects at each position are essentially independentof each other (i.e., independent binding of individual side-chains).When residue j occurs at position i in the peptide, it is assumed tocontribute a constant amount j, to the free energy of binding of thepeptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has beendescribed in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (seealso Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al.,J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchorand non-anchor alike, the geometric mean of the average relative binding(ARB) of all peptides carrying j is calculated relative to the remainderof the group, and used as the estimate of j_(i). For Class II peptides,if multiple alignments are possible, only the highest scoring alignmentis utilized, following an iterative procedure. To calculate an algorithmscore of a given peptide in a test set, the ARB values corresponding tothe sequence of the peptide are multiplied. If this product exceeds achosen threshold, the peptide is predicted to bind. Appropriatethresholds are chosen as a function of the degree of stringency ofprediction desired.

Selection of HLA-A2 Supertype Cross-Reactive Peptides

Protein sequences from 193P1E1B are scanned utilizing motifidentification software, to identify 8-, 9-10- and 11-mer sequencescontaining the HLA-A2-supermotif main anchor specificity. Typically,these sequences are then scored using the protocol described above andthe peptides corresponding to the positive-scoring sequences aresynthesized and tested for their capacity to bind purified HLA-A*0201molecules in vitro (HLA-A*0201 is considered a prototype A2 supertypemolecule).

These peptides are then tested for the capacity to bind to additionalA2-supertype molecules (A*0202, A*0203, A*0206, and A*6802). Peptidesthat bind to at least three of the five A2-supertype alleles tested aretypically deemed A2-supertype cross-reactive binders. Preferred peptidesbind at an affinity equal to or less than 500 nM to three or more HLA-A2supertype molecules.

Selection of HLA-A3 Supermotif-Bearing Epitopes

The 193P1E1B protein sequence(s) scanned above is also examined for thepresence of peptides with the HLA-A3-supermotif primary anchors.Peptides corresponding to the HLA A3 supermotif-bearing sequences arethen synthesized and tested for binding to HLA-A*0301 and HLA-A*1101molecules, the molecules encoded by the two most prevalent A3-supertypealleles. The peptides that bind at least one of the two alleles withbinding affinities of ≦500 nM, often ≦200 nM, are then tested forbinding cross-reactivity to the other common A3-supertype alleles (e.g.,A*3101, A*3301, and A*6801) to identify those that can bind at leastthree of the five HLA-A3-supertype molecules tested.

Selection of HLA-B7 supermotif bearing epitopes

The 193P1E1B protein(s) scanned above is also analyzed for the presenceof 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif.Corresponding peptides are synthesized and tested for binding toHLA-B*0702, the molecule encoded by the most common B7-supertype allele(i.e., the prototype B7 supertype allele). Peptides binding B*0702 withIC₅₀ of ≦500 nM are identified using standard methods. These peptidesare then tested for binding to other common B7-supertype molecules(e.g., B*3501, B*5101, B*5301, and B*5401). Peptides capable of bindingto three or more of the five B7-supertype alleles tested are therebyidentified.

Selection of A1 and A24 Motif-Bearing Epitopes

To further increase population coverage, HLA-A1 and -A24 epitopes canalso be incorporated into vaccine compositions. An analysis of the193P1E1B protein can also be performed to identify HLA-A1- andA24-motif-containing sequences.

High affinity and/or cross-reactive binding epitopes that bear othermotif and/or supermotifs are identified using analogous methodology.

Example 14 Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that areidentified as described herein are selected to confirm in vitroimmunogenicity. Confirmation is performed using the followingmethodology:

Target Cell Lines for Cellular Screening:

The .221A2.1 cell line, produced by transferring the HLA-A2.1 gene intothe HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221,is used as the peptide-loaded target to measure activity ofHLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 mediumsupplemented with antibiotics, sodium pyruvate, nonessential amino acidsand 10% (v/v) heat inactivated FCS. Cells that express an antigen ofinterest, or transfectants comprising the gene encoding the antigen ofinterest, can be used as target cells to confirm the ability ofpeptide-specific CTLs to recognize endogenous antigen.

Primary CTL Induction Cultures:

Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640plus 5% AB human serum, non-essential amino acids, sodium pyruvate,L-glutamine and penicillin/streptomycin). The monocytes are purified byplating 10×10⁶ PBMC/well in a 6-well plate. After 2 hours at 37° C., thenon-adherent cells are removed by gently shaking the plates andaspirating the supernatants. The wells are washed a total of three timeswith 3 ml RPMI to remove most of the non-adherent and loosely adherentcells. Three ml of complete medium containing 50 ng/ml of GM-CSF and1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCson day 6 at 75 ng/ml and the cells are used for CTL induction cultureson day 7.

Induction of CTL with DC and Peptide: CD8+ T-cells are isolated bypositive selection with Dynal immunomagnetic beads (Dynabeads® M450) andthe detacha-bead® reagent. Typically about 200-250×10⁶ PBMC areprocessed to obtain 24×10⁶ CD8, T-cells (enough for a 48-well plateculture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse,washed once with PBS containing 1% human AB serum and resuspended inPBS/1% AB serum at a concentration of 20×10⁶cells/ml. The magnetic beadsare washed 3 times with PBS/AB serum, added to the cells (140 μlbeads/20×10⁶ cells) and incubated for 1 hour at 4° C. with continuousmixing. The beads and cells are washed 4× with PBS/AB serum to removethe nonadherent cells and resuspended at 100×10⁶ cells/ml (based on theoriginal cell number) in PBS/AB serum containing 100 μl/ml detacha-bead®reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at roomtemperature with continuous mixing. The beads are washed again withPBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected andcentrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1%BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentrationof 1-2×10⁶/ml in the presence of 3 μg/ml 92-microglobulin for 4 hours at20° C. The DC are then irradiated (4,200 rads), washed 1 time withmedium and counted again.

Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×10⁵cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×10⁶cell/ml) in each well of a 48-well plate in the presence of 10 μg/ml ofIL-7. Recombinant human IL-10 is added the next day at a finalconcentration of 10 μg/ml and rhuman IL-2 is added 48 hours later at 10IU/ml.

Restimulation of the induction cultures with peptide-pulsed adherentcells: Seven and fourteen days after the primary induction, the cellsare restimulated with peptide-pulsed adherent cells. The PBMCs arethawed and washed twice with RPMI and DNAse. The cells are resuspendedat 5×10⁶ cells/ml and irradiated at −4200 rads. The PBMCs are plated at2×10⁶ in 0.5 ml complete medium per well and incubated for 2 hours at37° C. The plates are washed twice with RPMI by tapping the plate gentlyto remove the nonadherent cells and the adherent cells pulsed with 10μg/ml of peptide in the presence of 3 μg/ml 92 microglobulin in 0.25 mlRPMI/5% AB per well for 2 hours at 37° C. Peptide solution from eachwell is aspirated and the wells are washed once with RPMI. Most of themedia is aspirated from the induction cultures (CD8+ cells) and broughtto 0.5 ml with fresh media. The cells are then transferred to the wellscontaining the peptide-pulsed adherent cells. Twenty four hours laterrecombinant human IL-10 is added at a final concentration of 10 μg/mland recombinant human IL2 is added the next day and again 2-3 days laterat 50 UI/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75,1998). Seven days later, the cultures are assayed for CTL activity in a⁵¹Cr release assay. In some experiments the cultures are assayed forpeptide-specific recognition in the in situ IFNγ ELISA at the time ofthe second restimulation followed by assay of endogenous recognition 7days later. After expansion, activity is measured in both assays for aside-by-side comparison.

Measurement of CTL Lytic Activity by ⁵¹Cr Release.

Seven days after the second restimulation, cytotoxicity is determined ina standard (5 hr) ⁵¹Cr release assay by assaying individual wells at asingle E:T. Peptide-pulsed targets are prepared by incubating the cellswith 10 μg/ml peptide overnight at 37° C.

Adherent target cells are removed from culture flasks with trypsin-EDTA.Target cells are labeled with 200 μCi of ⁵¹Cr sodium chromate (Dupont,Wilmington, Del.) for 1 hour at 37° C. Labeled target cells areresuspended at 10⁶ per ml and diluted 1:10 with K562 cells at aconcentration of 3.3×10⁶/ml (an NK-sensitive erythroblastoma cell lineused to reduce non-specific lysis). Target cells (100 μl) and effectors(100 μl) are plated in 96 well round-bottom plates and incubated for 5hours at 37° C. At that time, 100 μl of supernatant are collected fromeach well and percent lysis is determined according to the formula:

[(cpm of the test sample-cpm of the spontaneous ⁵¹Cr releasesample)/(cpm of the maximal ⁵¹Cr release sample-cpm of the spontaneous⁵¹Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeledtargets with 1% Triton X-100 and media alone, respectively. A positiveculture is defined as one in which the specific lysis(sample-background) is 10% or higher in the case of individual wells andis 15% or more at the two highest E:T ratios when expanded cultures areassayed.

In Situ Measurement of Human IFNγ Production as an Indicator ofPeptide-Specific and Endogenous Recognition

Immulon 2 plates are coated with mouse anti-human IFNγ monoclonalantibody (4 μg/ml 0.1M NaHCO₃, pH8.2) overnight at 4° C. The plates arewashed with Ca²⁺, Mg²⁺ free PBS/0.05% Tween 20 and blocked with PBS/10%FCS for two hours, after which the CTLs (100 μl/well) and targets (100μl/well) are added to each well, leaving empty wells for the standardsand blanks (which received media only). The target cells, eitherpeptide-pulsed or endogenous targets, are used at a concentration of1×10⁶ cells/mi. The plates are incubated for 48 hours at 37° C. with 5%CO₂.

Recombinant human IFN-gamma is added to the standard wells starting at400 pg or 1200 pg/100 microliter/well and the plate incubated for twohours at 37° C. The plates are washed and 100 μl of biotinylated mouseanti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at roomtemperature. After washing again, 100 microliter HRP-streptavidin(1:4000) are added and the plates incubated for one hour at roomtemperature. The plates are then washed 6× with wash buffer, 100microliter/well developing solution (TMB 1:1) are added, and the platesallowed to develop for 5-15 minutes. The reaction is stopped with 50microliter/well 1M H₃PO₄ and read at OD450. A culture is consideredpositive if it measured at least 50 pg of IFN-gamma/well abovebackground and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity againstpeptide-pulsed targets and/or tumor targets are expanded over a two weekperiod with anti-CD3. Briefly, 5×10⁴ CD8+ cells are added to a T25 flaskcontaining the following: 1×10⁶ irradiated (4, 200 rad) PBMC (autologousor allogeneic) per ml, 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 25μM 2-mercaptoethanol, L-glutamine andpenicillin/streptomycin. Recombinant human IL2 is added 24 hours laterat a final concentration of 200 IU/ml and every three days thereafterwith fresh media at 50 IU/ml. The cells are split if the cellconcentration exceeds 1×10⁶/ml and the cultures are assayed between days13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assayor at 1×10⁶/ml in the in situ IFNγ assay using the same targets asbefore the expansion.

Cultures are expanded in the absence of anti-CD3, as follows. Thosecultures that demonstrate specific lytic activity against peptide andendogenous targets are selected and 5×10⁴ CD8+ cells are added to a T25flask containing the following: 1×10⁶ autologous PBMC per ml which havebeen peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. andirradiated (4,200 rad); 2×10⁵ irradiated (8,000 rad) EBV-transformedcells per ml RPMI-1640 containing 10% (v/v) human AB serum,non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine andgentamicin.

Immunogenicity of A2 Supermotif-Bearing Peptides

A2-supermotif cross-reactive binding peptides are tested in the cellularassay for the ability to induce peptide-specific CTL in normalindividuals. In this analysis, a peptide is typically considered to bean epitope if it induces peptide-specific CTLs in at least individuals,and preferably, also recognizes the endogenously expressed peptide.

Immunogenicity can also be confirmed using PBMCs isolated from patientsbearing a tumor that expresses 193P1E1B. Briefly, PBMCs are isolatedfrom patients, re-stimulated with peptide-pulsed monocytes and assayedfor the ability to recognize peptide-pulsed target cells as well astransfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 Immunogenicity

HLA-A3 supermotif-bearing cross-reactive binding peptides are alsoevaluated for immunogenicity using methodology analogous for that usedto evaluate the immunogenicity of the HLA-A2 supermotif peptides.

Evaluation of B7 Immunogenicity

Immunogenicity screening of the B7-supertype cross-reactive bindingpeptides identified as set forth herein are confirmed in a manneranalogous to the confirmation of A2-and A3-supermotif-bearing peptides.

Peptides bearing other supermotifs/motifs, e.g., HLA-A1, HLA-A24 etc.are also confirmed using similar methodology

Example 15 Implementation of the Extended Supermotif to Improve theBinding Capacity of Native Epitopes by Creating Analogs

HLA motifs and supermotifs (comprising primary and/or secondaryresidues) are useful in the identification and preparation of highlycross-reactive native peptides, as demonstrated herein. Moreover, thedefinition of HLA motifs and supermotifs also allows one to engineerhighly cross-reactive epitopes by identifying residues within a nativepeptide sequence which can be analoged to confer upon the peptidecertain characteristics, e.g. greater cross-reactivity within the groupof HLA molecules that comprise a supertype, and/or greater bindingaffinity for some or all of those HLA molecules. Examples of analogingpeptides to exhibit modulated binding affinity are set forth in thisexample.

Analoging at Primary Anchor Residues

Peptide engineering strategies are implemented to further increase thecross-reactivity of the epitopes. For example, the main anchors ofA2-supermotif-bearing peptides are altered, for example, to introduce apreferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineeredanalog is initially tested for binding to the prototype A2 supertypeallele A*0201, then, if A*0201 binding capacity is maintained, forA2-supertype cross-reactivity.

Alternatively, a peptide is confirmed as binding one or all supertypemembers and then analoged to modulate binding affinity to any one (ormore) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screeninganalysis is typically further restricted by the capacity of the parentwild type (WT) peptide to bind at least weakly, i.e., bind at an IC₅₀ of5000 nM or less, to three of more A2 supertype alleles. The rationalefor this requirement is that the WT peptides must be presentendogenously in sufficient quantity to be biologically relevant.Analoged peptides have been shown to have increased immunogenicity andcross-reactivity by T cells specific for the parent epitope (see, e.g.,Parkhurst et al., J. Immunol. 157:2539, 1996; and Pogue et al., Proc.Natl. Acad. Sci. USA 92:8166, 1995).

In the cellular screening of these peptide analogs, it is important toconfirm that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, target cells that endogenouslyexpress the epitope.

Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides

Analogs of HLA-A3 supermotif-bearing epitopes are generated usingstrategies similar to those employed in analoging HLA-A2supermotif-bearing peptides. For example, peptides binding to 3/5 of theA3-supertype molecules are engineered at primary anchor residues topossess a preferred residue (V, S, M, or A) at position 2.

The analog peptides are then tested for the ability to bind A*03 andA*11 (prototype A3 supertype alleles). Those peptides that demonstrate≦500 nM binding capacity are then confirmed as having A3-supertypecross-reactivity.

Similarly to the A2- and A3-motif bearing peptides, peptides binding 3or more B7-supertype alleles can be improved, where possible, to achieveincreased cross-reactive binding or greater binding affinity or bindinghalf life. B7 supermotif-bearing peptides are, for example, engineeredto possess a preferred residue (V, I, L, or F) at the C-terminal primaryanchor position, as demonstrated by Sidney et al. (J. Immunol.157:3480-3490, 1996).

Analoging at primary anchor residues of other motif and/orsupermotif-bearing epitopes is performed in a like manner.

The analog peptides are then be confirmed for immunogenicity, typicallyin a cellular screening assay. Again, it is generally important todemonstrate that analog-specific CTLs are also able to recognize thewild-type peptide and, when possible, targets that endogenously expressthe epitope.

Analoging at Secondary Anchor Residues

Moreover, HLA supermotifs are of value in engineering highlycross-reactive peptides and/or peptides that bind HLA molecules withincreased affinity by identifying particular residues at secondaryanchor positions that are associated with such properties. For example,the binding capacity of a B7 supermotif-bearing peptide with an Fresidue at position 1 is analyzed. The peptide is then analoged to, forexample, substitute L for F at position 1. The analoged peptide isevaluated for increased binding affinity, binding half life and/orincreased cross-reactivity. Such a procedure identifies analogedpeptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity orcross-reactivity can also be tested for immunogenicity inHLA-B7-transgenic mice, following for example, IFA immunization orlipopeptide immunization. Analoged peptides are additionally tested forthe ability to stimulate a recall response using PBMC from patients with193P1E1B-expressing tumors.

Other Analoging Strategies

Another form of peptide analoging, unrelated to anchor positions,involves the substitution of a cysteine with α-amino butyric acid. Dueto its chemical nature, cysteine has the propensity to form disulfidebridges and sufficiently alter the peptide structurally so as to reducebinding capacity. Substitution of α-amino butyric acid for cysteine notonly alleviates this problem, but has been shown to improve binding andcrossbinding capabilities in some instances (see, e.g., the review bySette et al., In: Persistent Viral Infections, Eds. R. Ahmed and 1.Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the bindingproperties and/or cross-reactivity of peptide ligands for HLA supertypemolecules can be modulated.

Example 16 Identification and Confirmation of 193P1E1B-Derived Sequenceswith HLA-DR Binding Motifs

Peptide epitopes bearing an HLA class II supermotif or motif areidentified and confirmed as outlined below using methodology similar tothat described for HLA Class I peptides.

Selection of HLA-DR-Supermotif-Bearing Epitopes.

To identify 193P1E1B-derived, HLA class II HTL epitopes, a 193P1E1Bantigen is analyzed for the presence of sequences bearing anHLA-DR-motif or supermotif. Specifically, 15-mer sequences are selectedcomprising a DR-supermotif, comprising a 9-mer core, add three-residueN- and C-terminal flanking regions (15 amino acids total).

Protocols for predicting peptide binding to DR molecules have beendeveloped (Southwood et al., J. Immunol. 160:3363-3373, 1998). Theseprotocols, specific for individual DR molecules, allow the scoring, andranking, of 9-mer core regions. Each protocol not only scores peptidesequences for the presence of DR-supermotif primary anchors (i.e., atposition 1 and position 6) within a 9-mer core, but additionallyevaluates sequences for the presence of secondary anchors. Usingallele-specific selection tables (see, e.g., Southwood et al., ibid.),it has been found that these protocols efficiently select peptidesequences with a high probability of binding a particular DR molecule.Additionally, it has been found that performing these protocols intandem, specifically those for DR1, DR4w4, and DR7, can efficientlyselect DR cross-reactive peptides.

The 193P1E1B-derived peptides identified above are tested for theirbinding capacity for various common HLA-DR molecules. All peptides areinitially tested for binding to the DR molecules in the primary panel:DR1, DR4w4, and DR7. Peptides binding at least two of these three DRmolecules are then tested for binding to DR2w2 β1, DR2w2 β2, DR6w19, andDR9 molecules in secondary assays. Finally, peptides binding at leasttwo of the four secondary panel DR molecules, and thus cumulatively atleast four of seven different DR molecules, are screened for binding toDR4w15, DR5w11, and DR8w2 molecules in tertiary assays. Peptides bindingat least seven of the ten DR molecules comprising the primary,secondary, and tertiary screening assays are considered cross-reactiveDR binders. 193P1E1B-derived peptides found to bind common HLA-DRalleles are of particular interest.

Selection of DR3 Motif Peptides

Because HLA-DR3 is an allele that is prevalent in Caucasian, Black, andHispanic populations, DR3 binding capacity is a relevant criterion inthe selection of HTL epitopes. Thus, peptides shown to be candidates mayalso be assayed for their DR3 binding capacity. However, in view of thebinding specificity of the DR3 motif, peptides binding only to DR3 canalso be considered as candidates for inclusion in a vaccine formulation.

To efficiently identify peptides that bind DR3, target 193P1E1B antigensare analyzed for sequences carrying one of the two DR3-specific bindingmotifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). Thecorresponding peptides are then synthesized and confirmed as having theability to bind DR3 with an affinity of 1 μM or better, i.e., less than1 μM. Peptides are found that meet this binding criterion and qualify asHLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccinecompositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the classII motif-bearing peptides are analoged to improve affinity orcross-reactivity. For example, aspartic acid at position 4 of the 9-mercore sequence is an optimal residue for DR3 binding, and substitutionfor that residue often improves DR 3 binding.

Example 17 Immunogenicity of 193P1E1B-Derived HTL Epitopes

This example determines immunogenic DR supermotif- and DR3 motif-bearingepitopes among those identified using the methodology set forth herein.

Immunogenicity of HTL epitopes are confirmed in a manner analogous tothe determination of immunogenicity of CTL epitopes, by assessing theability to stimulate HTL responses and/or by using appropriatetransgenic mouse models. Immunogenicity is determined by screening for:1.) in vitro primary induction using normal PBMC or 2.) recall responsesfrom patients who have 193P1E1B-expressing tumors.

Example 18 Calculation of Phenotypic Frequencies of HLA-Supertypes inVarious Ethnic Backgrounds to Determine Breadth of Population Coverage

This example illustrates the assessment of the breadth of populationcoverage of a vaccine composition comprised of multiple epitopescomprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA allelesare determined. Gene frequencies for each HLA allele are calculated fromantigen or allele frequencies utilizing the binomial distributionformulae gf=1-(SQRT(1-af)) (see, e.g., Sidney et al., Human Immunol.45:79-93, 1996). To obtain overall phenotypic frequencies, cumulativegene frequencies are calculated, and the cumulative antigen frequenciesderived by the use of the inverse formula [af=1-(1-Cgf)²].

Where frequency data is not available at the level of DNA typing,correspondence to the serologically defined antigen frequencies isassumed. To obtain total potential supertype population coverage nolinkage disequilibrium is assumed, and only alleles confirmed to belongto each of the supertypes are included (minimal estimates). Estimates oftotal potential coverage achieved by inter-loci combinations are made byadding to the A coverage the proportion of the non-A covered populationthat could be expected to be covered by the B alleles considered (e.g.,total=A+B*(1-A)). Confirmed members of the A3-like supertype are A3,A11, A31, A*3301, and A*6801. Although the A3-like supertype may alsoinclude A34, A66, and A*7401, these alleles were not included in overallfrequency calculations. Likewise, confirmed members of the A2-likesupertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206,A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmedalleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601,B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, andB*5602).

Population coverage achieved by combining the A2-, A3- and B7-supertypesis approximately 86% in five major ethnic groups. Coverage may beextended by including peptides bearing the A1 and A24 motifs. Onaverage, A1 is present in 12% and A24 in 29% of the population acrossfive different major ethnic groups (Caucasian, North American Black,Chinese, Japanese, and Hispanic). Together, these alleles arerepresented with an average frequency of 39% in these same ethnicpopulations. The total coverage across the major ethnicities when A1 andA24 are combined with the coverage of the A2-, A3- and B7-supertypealleles is >95%, see, e.g., Table IV (G). An analogous approach can beused to estimate population coverage achieved with combinations of classII motif-bearing epitopes.

Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest.100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al.,J. Immunol. 159:1648, 1997) have shown that highly cross-reactivebinding peptides are almost always recognized as epitopes. The use ofhighly cross-reactive binding peptides is an important selectioncriterion in identifying candidate epitopes for inclusion in a vaccinethat is immunogenic in a diverse population.

With a sufficient number of epitopes (as disclosed herein and from theart), an average population coverage is predicted to be greater than 95%in each of five major ethnic populations. The game theory Monte Carlosimulation analysis, which is known in the art (see e.g., Osborne, M. J.and Rubinstein, A. “A course in game theory” MIT Press, 1994), can beused to estimate what percentage of the individuals in a populationcomprised of the Caucasian, North American Black, Japanese, Chinese, andHispanic ethnic groups would recognize the vaccine epitopes describedherein. A preferred percentage is 90%. A more preferred percentage is95%.

Example 19 CTL Recognition Of Endogenously Processed Antigens AfterPriming

This example confirms that CTL induced by native or analoged peptideepitopes identified and selected as described herein recognizeendogenously synthesized, i.e., native antigens.

Effector cells isolated from transgenic mice that are immunized withpeptide epitopes, for example HLA-A2 supermotif-bearing epitopes, arere-stimulated in vitro using peptide-coated stimulator cells. Six dayslater, effector cells are assayed for cytotoxicity and the cell linesthat contain peptide-specific cytotoxic activity are furtherre-stimulated. An additional six days later, these cell lines are testedfor cytotoxic activity on ⁵¹Cr labeled Jurkat-A2.l/K^(b) target cells inthe absence or presence of peptide, and also tested on ⁵¹Cr labeledtarget cells bearing the endogenously synthesized antigen, i.e. cellsthat are stably transfected with 193P1E1B expression vectors.

The results demonstrate that CTL lines obtained from animals primed withpeptide epitope recognize endogenously synthesized 193P1E1B antigen. Thechoice of transgenic mouse model to be used for such an analysis dependsupon the epitope(s) that are being evaluated. In addition toHLA-A*0201/K^(b) transgenic mice, several other transgenic mouse modelsincluding mice with human A11, which may also be used to evaluate A3epitopes, and B7 alleles have been characterized and others (e.g.,transgenic mice for HLA-A1 and A24) are being developed. HLA-DR1 andHLA-DR3 mouse models have also been developed, which may be used toevaluate HTL epitopes.

Example 20 Activity Of CTL-HTL Conjugated Epitopes In Transgenic Mice

This example illustrates the induction of CTLs and HTLs in transgenicmice, by use of a 193P1E1B-derived CTL and HTL peptide vaccinecompositions. The vaccine composition used herein comprise peptides tobe administered to a patient with a 193P1E1B-expressing tumor. Thepeptide composition can comprise multiple CTL and/or HTL epitopes. Theepitopes are identified using methodology as described herein. Thisexample also illustrates that enhanced immunogenicity can be achieved byinclusion of one or more HTL epitopes in a CTL vaccine composition; sucha peptide composition can comprise an HTL epitope conjugated to a CTLepitope. The CTL epitope can be one that binds to multiple HLA familymembers at an affinity of 500 nM or less, or analogs of that epitope.The peptides may be lipidated, if desired.

Immunization procedures: Immunization of transgenic mice is performed asdescribed (Alexander et al., J. Immunol. 159:4753-4761, 1997). Forexample, A2/K^(b) mice, which are transgenic for the human HLA A2.1allele and are used to confirm the immunogenicity of HLA-A*0201 motif-or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously(base of the tail) with a 0.1 ml of peptide in Incomplete Freund'sAdjuvant, or if the peptide composition is a lipidated CTL/HTLconjugate, in DMSO/saline, or if the peptide composition is apolypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days afterpriming, splenocytes obtained from these animals are restimulated withsyngenic irradiated LPS-activated lymphoblasts coated with peptide.

Cell lines: Target cells for peptide-specific cytotoxicity assays areJurkat cells transfected with the HLA-A2.1/K^(b) chimeric gene (e.g.,Vitiello et at, J. Exp. Med. 173:1007, 1991)

In vitro CTL activation: One week after priming, spleen cells (30×10⁶cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000rads), peptide coated lymphoblasts (10×10⁶ cells/flask) in 10 ml ofculture medium/T25 flask. After six days, effector cells are harvestedand assayed for cytotoxic activity.

Assay for cytotoxic activity: Target cells (1.0 to 1.5×10⁶) areincubated at 37° C. in the presence of 200 μl of ⁵¹Cr. After 60 minutes,cells are washed three times and resuspended in R10 medium. Peptide isadded where required at a concentration of 1 μg/ml. For the assay, 10⁴⁵¹Cr-labeled target cells are added to different concentrations ofeffector cells (final volume of 200 μl) in U-bottom 96-well plates.After a six hour incubation period at 37° C., a 0.1 ml aliquot ofsupernatant is removed from each well and radioactivity is determined ina Micromedic automatic gamma counter. The percent specific lysis isdetermined by the formula: percent specific release=100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release). Tofacilitate comparison between separate CTL assays run under the sameconditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells.One lytic unit is arbitrarily defined as the number of effector cellsrequired to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Crrelease assay. To obtain specific lytic units/10⁶, the lytic units/10⁶obtained in the absence of peptide is subtracted from the lyticunits/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Crrelease is obtained at the effector (E): target (T) ratio of 50:1 (i.e.,5×10⁵ effector cells for 10,000 targets) in the absence of peptide and5:1 (i.e., 5×10⁴ effector cells for 10,000 targets) in the presence ofpeptide, the specific lytic units would be:[(1/50,000)-(1/500,000)]×10⁶=18 LU.

The results are analyzed to assess the magnitude of the CTL responses ofanimals injected with the immunogenic CTL/HTL conjugate vaccinepreparation and are compared to the magnitude of the CTL responseachieved using, for example, CTL epitopes as outlined above in theExample entitled “Confirmation of Immunogenicity.” Analyses similar tothis may be performed to confirm the immunogenicity of peptideconjugates containing multiple CTL epitopes and/or multiple HTLepitopes. In accordance with these procedures, it is found that a CTLresponse is induced, and concomitantly that an HTL response is inducedupon administration of such compositions.

Example 21 Selection of CTL and HTL Epitopes for Inclusion in a193P1E1B-Specific Vaccine.

This example illustrates a procedure for selecting peptide epitopes forvaccine compositions of the invention. The peptides in the compositioncan be in the form of a nucleic acid sequence, either single or one ormore sequences (i.e., minigene) that encodes peptide(s), or can besingle and/or polyepitopic peptides.

The following principles are utilized when selecting a plurality ofepitopes for inclusion in a vaccine composition. Each of the followingprinciples is balanced in order to make the selection.

Epitopes are selected which, upon administration, mimic immune responsesthat are correlated with 193P1E1B clearance. The number of epitopes useddepends on observations of patients who spontaneously clear 193P1E1B.For example, if it has been observed that patients who spontaneouslyclear 193P1E1B-expressing cells generate an immune response to at leastthree (3) epitopes from 193P1E1B antigen, then at least three epitopesshould be included for HLA class I. A similar rationale is used todetermine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an IC₅₀ of500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of1000 nM or less; or HLA Class I peptides with high binding scores fromthe BIMAS web site.

In order to achieve broad coverage of the vaccine through out a diversepopulation, sufficient supermotif bearing peptides, or a sufficientarray of allele-specific motif bearing peptides, are selected to givebroad population coverage. In one embodiment, epitopes are selected toprovide at least 80% population coverage. A Monte Carlo analysis, astatistical evaluation known in the art, can be employed to assessbreadth, or redundancy, of population coverage.

When creating polyepitopic compositions, or a minigene that encodessame, it is typically desirable to generate the smallest peptidepossible that encompasses the epitopes of interest. The principlesemployed are similar, if not the same, as those employed when selectinga peptide comprising nested epitopes. For example, a protein sequencefor the vaccine composition is selected because it has maximal number ofepitopes contained within the sequence, i.e., it has a highconcentration of epitopes. Epitopes may be nested or overlapping (i.e.,frame shifted relative to one another). For example, with overlappingepitopes, two 9-mer epitopes and one 10-mer epitope can be present in a10 amino acid peptide. Each epitope can be exposed and bound by an HLAmolecule upon administration of such a peptide. A multi-epitopic,peptide can be generated synthetically, recombinantly, or via cleavagefrom the native source. Alternatively, an analog can be made of thisnative sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes. This embodimentprovides for the possibility that an as yet undiscovered aspect ofimmune system processing will apply to the native nested sequence andthereby facilitate the production of therapeutic or prophylactic immuneresponse-inducing vaccine compositions. Additionally such an embodimentprovides for the possibility of motif-bearing epitopes for an HLA makeupthat is presently unknown. Furthermore, this embodiment (absent thecreating of any analogs) directs the immune response to multiple peptidesequences that are actually present in 193P1E1B, thus avoiding the needto evaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing nucleic acid vaccine compositions.Related to this embodiment, computer programs can be derived inaccordance with principles in the art, which identify in a targetsequence, the greatest number of epitopes per sequence length.

A vaccine composition comprised of selected, peptides, whenadministered, is safe, efficacious, and elicits an immune responsesimilar in magnitude to an immune response that controls or clears cellsthat bear or overexpress 193P1E1B.

Example 22 Construction of “Minigene” Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expressionplasmid. Minigene plasmids may, of course, contain variousconfigurations of B cell, CTL and/or HTL epitopes or epitope analogs asdescribed herein.

A minigene expression plasmid typically includes multiple CTL and HTLpeptide epitopes. In the present example, HLA-A2, -A3, -B7supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearingpeptide epitopes are used in conjunction with DR supenmotif-bearingepitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearingpeptide epitopes derived 193P1E1B, are selected such that multiplesupermotifs/motifs are represented to ensure broad population coverage.Similarly, HLA class II epitopes are selected from 193P1E1B to providebroad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearingepitopes and HLA DR-3 motif-bearing epitopes are selected for inclusionin the minigene construct. The selected CTL and HTL epitopes are thenincorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTLepitopes to the endoplasmic reticulum. For example, the li protein maybe fused to one or more HTL epitopes as described in the art, whereinthe CLIP sequence of the li protein is removed and replaced with an HLAclass II epitope sequence so that HLA class II epitope is directed tothe endoplasmic reticulum, where the epitope binds to an HLA class IImolecules.

This example illustrates the methods to be used for construction of aminigene-bearing expression plasmid. Other expression vectors that maybe used for minigene compositions are available and known to those ofskill in the art.

The minigene DNA plasmid of this example contains a consensus Kozaksequence and a consensus murine kappa Ig-light chain signal sequencefollowed by CTL and/or HTL epitopes selected in accordance withprinciples disclosed herein. The sequence encodes an open reading framefused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70nucleotides in length with 15 nucleotide overlaps, are synthesized andHPLC-purified. The oligonucleotides encode the selected peptide epitopesas well as appropriate linker nucleotides, Kozak sequence, and signalsequence. The final multiepitope minigene is assembled by extending theoverlapping oligonucleotides in three sets of reactions using PCR. APerkin/Elmer 9600 PCR machine is used and a total of 30 cycles areperformed using the following conditions: 95° C. for 15 sec, annealingtemperature (5° below the lowest calculated Tm of each primer pair) for30 sec, and 72° C. for 1 min.

For example, a minigene is prepared as follows. For a first PCRreaction, 5 μg of each of two oligonucleotides are annealed andextended: In an example using eight oligonucleotides, i.e., four pairsof primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100μl reactions containing Pfu polymerase buffer (1×=10 mM KCL, 10 mM(NH4)₂SO₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO₄, 0.1% Triton X-100,100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. Thefull-length dimer products are gel-purified, and two reactionscontaining the product of 1+2 and 3+4, and the product of 5+6 and 7+8are mixed, annealed, and extended for 10 cycles. Half of the tworeactions are then mixed, and 5 cycles of annealing and extensioncarried out before flanking primers are added to amplify the full lengthproduct. The full-length product is gel-purified and cloned intopCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 23 The Plasmid Construct and the Degree to Which It InducesImmunogenicity.

The degree to which a plasmid construct, for example a plasmidconstructed in accordance with the previous Example, is able to induceimmunogenicity is confirmed in vitro by determining epitope presentationby APC following transduction or transfection of the APC with anepitope-expressing nucleic acid construct. Such a study determines“antigenicity” and allows the use of human APC. The assay determines theability of the epitope to be presented by the APC in a context that isrecognized by a T cell by quantifying the density of epitope-HLA class Icomplexes on the cell surface. Quantitation can be performed by directlymeasuring the amount of peptide eluted from the APC (see, e.g., Sijts etal., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684,1989); or the number of peptide-HLA class I complexes can be estimatedby measuring the amount of lysis or lymphokine release induced bydiseased or transfected target cells, and then determining theconcentration of peptide necessary to obtain equivalent levels of lysisor lymphokine release (see, e.g., Kageyama et al., J. Immunol.154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injectionsinto mice and subsequent in vitro assessment of CTL and HTL activity,which are analyzed using cytotoxicity and proliferation assays,respectively, as detailed e.g., in Alexander et al., Immunity 1:751-761,1994.

For example, to confirm the capacity of a DNA minigene constructcontaining at least one HLA-A2 supermotif peptide to induce CTLs invivo, HLA-A2.1/K^(b) transgenic mice, for example, are immunizedintramuscularly with 100 μg of naked cDNA. As a means of comparing thelevel of CTLs induced by cDNA immunization, a control group of animalsis also immunized with an actual peptide composition that comprisesmultiple epitopes synthesized as a single polypeptide as they would beencoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of therespective compositions (peptide epitopes encoded in the minigene or thepolyepitopic peptide), then assayed for peptide-specific cytotoxicactivity in a ⁵¹Cr release assay. The results indicate the magnitude ofthe CTL response directed against the A2-restricted epitope, thusindicating the in vivo immunogenicity of the minigene vaccine andpolyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responsesdirected toward the HLA-A2 supermotif peptide epitopes as does thepolyepitopic peptide vaccine. A similar analysis is also performed usingother HLA-A3 and HLA-B7 transgenic mouse models to assess CTL inductionby HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby it is alsofound that the minigene elicits appropriate immune responses directedtoward the provided epitopes.

To confirm the capacity of a class II epitope-encoding minigene toinduce HTLs in vivo, DR transgenic mice, or for those epitopes thatcross react with the appropriate mouse MHC molecule, I-A^(b)-restrictedmice, for example, are immunized intramuscularly with 100 μg of plasmidDNA. As a means of comparing the level of HTLs induced by DNAimmunization, a group of control animals is also immunized with anactual peptide composition emulsified in complete Freund's adjuvant.CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunizedanimals and stimulated with each of the respective compositions(peptides encoded in the minigene). The HTL response is measured using a³H-thymidine incorporation proliferation assay, (see, e.g., Alexander etal. Immunity 1:751-761, 1994). The results indicate the magnitude of theHTL response, thus demonstrating the in vivo immunogenicity of theminigene.

DNA minigenes, constructed as described in the previous Example, canalso be confirmed as a vaccine in combination with a boosting agentusing a prime boost protocol. The boosting agent can consist ofrecombinant protein (e.g., Bamelt et al., Aids Res. and HumanRetrovinises 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia,for example, expressing a minigene or DNA encoding the complete proteinof interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegahet al., Proc. Natl. Acad. Sci USA 95:7648-53, 1998; Hanke and McMichael,Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med.5:526-34, 1999).

For example, the efficacy of the DNA minigene used in a prime boostprotocol is initially evaluated in transgenic mice. In this example,A2.1/K^(b) transgenic mice are immunized IM with 100 μg of a DNAminigene encoding the immunogenic peptides including at least one HLA-A2supermotif-bearing peptide. After an incubation period (ranging from 3-9weeks), the mice are boosted IP with 10⁷pfu/mouse of a recombinantvaccinia virus expressing the same sequence encoded by the DNA minigene.Control mice are immunized with 100 μg of DNA or recombinant vacciniawithout the minigene sequence, or with DNA encoding the minigene, butwithout the vaccinia boost. After an additional incubation period of twoweeks, splenocytes from the mice are immediately assayed forpeptide-specific activity in an ELISPOT assay. Additionally, splenocytesare stimulated in vitro with the A2-restricted peptide epitopes encodedin the minigene and recombinant vaccinia, then assayed forpeptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicitsgreater immune responses toward the HLA-A2 supermotif peptides than withDNA alone. Such an analysis can also be performed using HLA-A11 orHLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 orHLA-B7 motif or supermotif epitopes. The use of prime boost protocols inhumans is described below in the Example entitled “Induction of CTLResponses Using a Prime Boost Protocol.”

Example 24 Peptide Compositions for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent193P1E1B expression in persons who are at risk for tumors that bear thisantigen. For example, a polyepitopic peptide epitope composition (or anucleic acid comprising the same) containing multiple CTL and HTLepitopes such as those selected in the above Examples, which are alsoselected to target greater than 80% of the population, is administeredto individuals at risk for a 193P1E1B-associated tumor.

For example, a peptide-based composition is provided as a singlepolypeptide that encompasses multiple epitopes. The vaccine is typicallyadministered in a physiological solution that comprises an adjuvant,such as Incomplete Freunds Adjuvant. The dose of peptide for the initialimmunization is from about 1 to about 50,000 μg, generally 100-5,000 μg,for a 70 kg patient. The initial administration of vaccine is followedby booster dosages at 4 weeks followed by evaluation of the magnitude ofthe immune response in the patient, by techniques that determine thepresence of epitope-specific CTL populations in a PBMC sample.Additional booster doses are administered as required. The compositionis found to be both safe and efficacious as a prophylaxis against193P1E1B-associated disease.

Alternatively, a composition typically comprising transfecting agents isused for the administration of a nucleic acid-based vaccine inaccordance with methodologies known in the art and disclosed herein.

Example 25 Polyepitopic Vaccine Compositions Derived from Native193P1E1B Sequences

A native 193P1E1B polyprotein sequence is analyzed, preferably usingcomputer algorithms defined for each class I and/or class II supermotifor motif, to identify “relatively short” regions of the polyprotein thatcomprise multiple epitopes. The “relatively short” regions arepreferably less in length than an entire native antigen. This relativelyshort sequence that contains multiple distinct or overlapping, “nested”epitopes can be used to generate a minigene construct. The construct isengineered to express the peptide, which corresponds to the nativeprotein sequence. The “relatively short” peptide is generally less than250 amino acids in length, often less than 100 amino acids in length,preferably less than 75 amino acids in length, and more preferably lessthan 50 amino acids in length. The protein sequence of the vaccinecomposition is selected because it has maximal number of epitopescontained within the sequence, i.e., it has a high concentration ofepitopes. As noted herein, epitope motifs may be nested or overlapping(i.e., frame shifted relative to one another). For example, withoverlapping epitopes, two 9-mer epitopes and one 10-mer epitope can bepresent in a 10 amino acid peptide. Such a vaccine composition isadministered for therapeutic or prophylactic purposes.

The vaccine composition will include, for example, multiple CTL epitopesfrom 193P1E1B antigen and at least one HTL epitope. This polyepitopicnative sequence is administered either as a peptide or as a nucleic acidsequence which encodes the peptide. Alternatively, an analog can be madeof this native sequence, whereby one or more of the epitopes comprisesubstitutions that alter the cross-reactivity and/or binding affinityproperties of the polyepitopic peptide.

The embodiment of this example provides for the possibility that an asyet undiscovered aspect of immune system processing will apply to thenative nested sequence and thereby facilitate the production oftherapeutic or prophylactic immune response-inducing vaccinecompositions. Additionally, such an embodiment provides for thepossibility of motif-bearing epitopes for an HLA makeup(s) that ispresently unknown. Furthermore, this embodiment (excluding an analogedembodiment) directs the immune response to multiple peptide sequencesthat are actually present in native 193P1E1B, thus avoiding the need toevaluate any junctional epitopes. Lastly, the embodiment provides aneconomy of scale when producing peptide or nucleic acid vaccinecompositions.

Related to this embodiment, computer programs are available in the artwhich can be used to identify in a target sequence, the greatest numberof epitopes per sequence length.

Example 26 Polyepitopic Vaccine Compositions from Multiple Antigens

The 193P1E1B peptide epitopes of the present invention are used inconjunction with epitopes from other target tumor-associated antigens,to create a vaccine composition that is useful for the prevention ortreatment of cancer that expresses 193P1E1B and such other antigens. Forexample, a vaccine composition can be provided as a single polypeptidethat incorporates multiple epitopes from 193P1E1B as well astumor-associated antigens that are often expressed with a target cancerassociated with 193P1E1B expression, or can be administered as acomposition comprising a cocktail of one or more discrete epitopes.Alternatively, the vaccine can be administered as a minigene constructor as dendritic cells which have been loaded with the peptide epitopesin vitro.

Example 27 Use of Peptides to Evaluate an Immune Response

Peptides of the invention may be used to analyze an immune response forthe presence of specific antibodies, CTL or HTL directed to 193P1E1B.Such an analysis can be performed in a manner described by Ogg et al.,Science 279:2103-2106, 1998. In this Example, peptides in accordancewith the invention are used as a reagent for diagnostic or prognosticpurposes, not as an immunogen.

In this example highly sensitive human leukocyte antigen tetramericcomplexes (“tetramers”) are used for a cross-sectional analysis of, forexample, 193P1E1B HLA-A*0201 -specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or followingimmunization comprising a 193P1E1B peptide containing an A*0201 motif.Tetrameric complexes are synthesized as described (Musey et al., N.Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201in this example) and P2-microglobulin are synthesized by means of aprokaryotic expression system. The heavy chain is modified by deletionof the transmembrane-cytosolic tail and COOH-terminal addition of asequence containing a BirA enzymatic biotinylation site. The heavychain, β2-microglobulin, and peptide are refolded by dilution. The 45-kDrefolded product is isolated by fast protein liquid chromatography andthen biotinylated by BirA in the presence of biotin (Sigma, St. Louis,Mo.), adenosine 5′ triphosphate and magnesium.Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, andthe tetrameric product is concentrated to 1 mg/ml. The resulting productis referred to as tetramer-phycoerythrin.

For the analysis of patient blood samples, approximately one millionPBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl ofcold phosphate-buffered saline. Tri-color analysis is performed with thetetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. ThePBMCs are incubated with tetramer and antibodies on ice for 30 to 60 minand then washed twice before formaldehyde fixation. Gates are applied tocontain >99.98% of control samples. Controls for the tetramers includeboth A*0201-negative individuals and A*0201-positive non-diseaseddonors. The percentage of cells stained with the tetramer is thendetermined by flow cytometry. The results indicate the number of cellsin the PBMC sample that contain epitope-restricted CTLs, thereby readilyindicating the extent of immune response to the 193P1E1B epitope, andthus the status of exposure to 193P1E1B, or exposure to a vaccine thatelicits a protective or therapeutic response.

Example 28 Use of Peptide Epitopes to Evaluate Recall Responses

The peptide epitopes of the invention are used as reagents to evaluate Tcell responses, such as acute or recall responses, in patients. Such ananalysis may be performed on patients who have recovered from193P1E1B-associated disease or who have been vaccinated with a 193P1E1Bvaccine.

For example, the class I restricted CTL response of persons who havebeen vaccinated may be analyzed. The vaccine may be any 193P1E1Bvaccine. PBMC are collected from vaccinated individuals and HLA typed.Appropriate peptide epitopes of the invention that, optimally, bearsupermotifs to provide cross-reactivity with multiple HLA supertypefamily members, are then used for analysis of samples derived fromindividuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaquedensity gradients (Sigma Chemical Co., St. Louis, Mo.), washed threetimes in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCOLaboratories) supplemented with L-glutamine (2mM), penicillin (50U/ml),streptomycin (50 μg/ml), and Hepes (10 mM) containing 10%heat-inactivated human AB serum (complete RPMI) and plated usingmicroculture formats. A synthetic peptide comprising an epitope of theinvention is added at 10 μg/ml to each well and HBV core 128-140 epitopeis added at 1 μg/ml to each well as a source of T cell help during thefirst week of stimulation.

In the microculture format, 4×10⁵ PBMC are stimulated with peptide in 8replicate cultures in 96-well round bottom plate in 100 II/well ofcomplete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/mlfinal concentration of rIL-2 are added to each well. On day 7 thecultures are transferred into a 96-well flat-bottom plate andrestimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad)autologous feeder cells. The cultures are tested for cytotoxic activityon day 14. A positive CTL response requires two or more of the eightreplicate cultures to display greater than 10% specific ⁵¹Cr release,based on comparison with non-diseased control subjects as previouslydescribed (Rehermann, et al., Nature Med. 2:1104, 1108, 1996; Rehermannet al., J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J.Clin. Invest. 98:1432-1440, 1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCLthat are either purchased from the American Society forHistocompatibility and Immunogenetics (ASHI, Boston, Mass.) orestablished from the pool of patients as described (Guilhot, et al. J.Virol. 66:2670-2678, 1992).

Cytotoxicity assays are performed in the following manner. Target cellsconsist of either allogeneic HLA-matched or autologous EBV-transformed Blymphoblastoid cell line that are incubated overnight with the syntheticpeptide epitope of the invention at 10 μM, and labeled with 100 μCi of⁵¹Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after whichthey are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well ⁵¹Crrelease assay using U-bottomed 96 well plates containing 3,000targets/well. Stimulated PBMC are tested at effector/target (E/T) ratiosof 20-50:1 on day 14. Percent cytotoxicity is determined from theformula: 100×[(experimental release-spontaneous release)/maximumrelease-spontaneous release)]. Maximum release is determined by lysis oftargets by detergent (2% Triton X-100; Sigma Chemical Co., St. Louis,Mo.). Spontaneous release is <25% of maximum release for allexperiments.

The results of such an analysis indicate the extent to whichHLA-restricted CTL populations have been stimulated by previous exposureto 193P1E1B or a 193P1E1B vaccine.

Similarly, Class II restricted HTL responses may also be analyzed.Purified PBMC are cultured in a 96-well flat bottom plate at a densityof 1.5×10⁵ cells/well and are stimulated with 10 μg/ml synthetic peptideof the invention, whole 193P1E1B antigen, or PHA. Cells are routinelyplated in replicates of 4-6 wells for each condition. After seven daysof culture, the medium is removed and replaced with fresh mediumcontaining 10 U/ml IL-2. Two days later, 1 μCi ³H-thymidine is added toeach well and incubation is continued for an additional 18 hours.Cellular DNA is then harvested on glass fiber mats and analyzed for³H-thymidine incorporation. Antigen-specific T cell proliferation iscalculated as the ratio of ³H-thymidine incorporation in the presence ofantigen divided by the ³H-thymidine incorporation in the absence ofantigen.

Example 29 Induction Of Specific CTL Response In Humans

A human clinical trial for an immunogenic composition comprising CTL andHTL epitopes of the invention is set up as an IND Phase I, doseescalation study and carried out as a randomized, double-blind,placebo-controlled trial. Such a trial is designed, for example, asfollows:

A total of about 27 individuals are enrolled and divided into 3 groups:

Group I: 3 subjects are injected with placebo and 6 subjects areinjected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects areinjected with 50 μg peptide composition;

Group III: 3 subjects are injected with placebo and 6 subjects areinjected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive abooster inoculation at the same dosage.

The endpoints measured in this study relate to the safety andtolerability of the peptide composition as well as its immunogenicity.Cellular immune responses to the peptide composition are an index of theintrinsic activity of this the peptide composition, and can therefore beviewed as a measure of biological efficacy. The following summarize theclinical and laboratory data that relate to safety and efficacyendpoints.

Safety: The incidence of adverse events is monitored in the placebo anddrug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy,subjects are bled before and after injection. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

The vaccine is found to be both safe and efficacious.

Example 30 Phase II Trials In Patients Expressing 193P1E1B

Phase II trials are performed to study the effect of administering theCTL-HTL peptide compositions to patients having cancer that expresses193P1E1B. The main objectives of the trial are to determine an effectivedose and regimen for inducing CTLs in cancer patients that express193P1E1B, to establish the safety of inducing a CTL and HTL response inthese patients, and to see to what extent activation of CTLs improvesthe clinical picture of these patients, as manifested, e.g., by thereduction and/or shrinking of lesions. Such a study is designed, forexample, as follows:

The studies are performed in multiple centers. The trial design is anopen-label, uncontrolled, dose escalation protocol wherein the peptidecomposition is administered as a single dose followed six weeks later bya single booster shot of the same dose. The dosages are 50, 500 and5,000 micrograms per injection. Drug-associated adverse effects(severity and reversibility) are recorded.

There are three patient groupings. The first group is injected with 50micrograms of the peptide composition and the second and third groupswith 500 and 5,000 micrograms of peptide composition, respectively. Thepatients within each group range in age from 21-65 and represent diverseethnic backgrounds. All of them have a tumor that expresses 193P1E1B.

Clinical manifestations or antigen-specific T-cell responses aremonitored to assess the effects of administering the peptidecompositions. The vaccine composition is found to be both safe andefficacious in the treatment of 193P1E1B-associated disease.

Example 31 Induction of CTL Responses Using a Prime Boost Protocol

A prime boost protocol similar in its underlying principle to that usedto confirm the efficacy of a DNA vaccine in transgenic mice, such asdescribed above in the Example entitled “The Plasmid Construct and theDegree to Which It Induces Immunogenicity,” can also be used for theadministration of the vaccine to humans. Such a vaccine regimen caninclude an initial administration of, for example, naked DNA followed bya boost using recombinant virus encoding the vaccine, or recombinantprotein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization may be performed using anexpression vector, such as that constructed in the Example entitled“Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form ofnaked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also beadministered using a gene gun. Following an incubation period of 3-4weeks, a booster dose is then administered. The booster can berecombinant fowlpox virus administered at a dose of 5-10⁷ to 5×10⁹ pfu.An alternative recombinant virus, such as an MVA, canarypox, adenovirus,or adeno-associated virus, can also be used for the booster, or thepolyepitopic protein or a mixture of the peptides can be administered.For evaluation of vaccine efficacy, patient blood samples are obtainedbefore immunization as well as at intervals following administration ofthe initial vaccine and booster doses of the vaccine. Peripheral bloodmononuclear cells are isolated from fresh heparinized blood byFicoll-Hypaque density gradient centrifugation, aliquoted in freezingmedia and stored frozen. Samples are assayed for CTL and HTL activity.

Analysis of the results indicates that a magnitude of responsesufficient to achieve a therapeutic or protective immunity against193P1E1B is generated.

Example 32 Administration of Vaccine Compositions Using Dendritic Cells(DC)

Vaccines comprising peptide epitopes of the invention can beadministered using APCs, or “professional” APCs such as DC. In thisexample, peptide-pulsed DC are administered to a patient to stimulate aCTL response in vivo. In this method, dendritic cells are isolated,expanded, and pulsed with a vaccine comprising peptide CTL and HTLepitopes of the invention. The dendritic cells are infused back into thepatient to elicit CTL and HTL responses in vivo. The induced CTL and HTLthen destroy or facilitate destruction, respectively, of the targetcells that bear the 193P1E1B protein from which the epitopes in thevaccine are derived.

For example, a cocktail of epitope-comprising peptides is administeredex vivo to PBMC, or isolated DC therefrom. A pharmaceutical tofacilitate harvesting of DC can be used, such as Progenipoietin™(Monsanto, St. Louis, Mo.) or GM-CSF/IL-4. After pulsing the DC withpeptides, and prior to reinfusion into patients, the DC are washed toremove unbound peptides.

As appreciated clinically, and readily determined by one of skill basedon clinical outcomes, the number of DC reinfused into the patient canvary (see, e.g., Nature Med. 4:328, 1998; Nature Med. 2:52, 1996 andProstate 32:272, 1997). Although 2-50×10⁶ DC per patient are typicallyadministered, larger number of DC, such as 10⁷ or 10⁸ can also beprovided. Such cell populations typically contain between 50-90% DC.

In some embodiments, peptide-loaded PBMC are injected into patientswithout purification of the DC. For example, PBMC generated aftertreatment with an agent such as Progenipoietin™ are injected intopatients without purification of the DC. The total number of PBMC thatare administered often ranges from 10⁸ to 10¹⁰. Generally, the celldoses injected into patients is based on the percentage of DC in theblood of each patient, as determined, for example, by immunofluorescenceanalysis with specific anti-DC antibodies. Thus, for example, ifProgenipoietin™ mobilizes 2% DC in the peripheral blood of a givenpatient, and that patient is to receive 5×10⁶ DC, then the patient willbe injected with a total of 2.5×10⁸ peptide-loaded PBMC. The percent DCmobilized by an agent such as Progenipoietin™ is typically estimated tobe between 2-10%, but can vary as appreciated by one of skill in theart.

Ex Vivo Activation of CTL/HTL Responses

Alternatively, ex vivo CTL or HTL responses to 193P1E1B antigens can beinduced by incubating, in tissue culture, the patient's, or geneticallycompatible, CTL or HTL precursor cells together with a source of APC,such as DC, and immunogenic peptides. After an appropriate incubationtime (typically about 7-28 days), in which the precursor cells areactivated and expanded into effector cells, the cells are infused intothe patient, where they will destroy (CTL) or facilitate destruction(HTL) of their specific target cells, i.e., tumor cells.

Example 33 An Alternative Method of Identifying and ConfirmingMotif-Bearing Peptides

Another method of identifying and confirming motif-bearing peptides isto elute them from cells bearing defined MHC molecules. For example, EBVtransformed B cell lines used for tissue typing have been extensivelycharacterized to determine which HLA molecules they express. In certaincases these cells express only a single type of HLA molecule. Thesecells can be transfected with nucleic acids that express the antigen ofinterest, e.g. 193P1E1B. Peptides produced by endogenous antigenprocessing of peptides produced as a result of transfection will thenbind to HLA molecules within the cell and be transported and displayedon the cell's surface. Peptides are then eluted from the HLA moleculesby exposure to mild acid conditions and their amino acid sequencedetermined, e.g., by mass spectral analysis (e.g., Kubo et at., J.Immunol. 152:3913, 1994). Because the majority of peptides that bind aparticular HLA molecule are motif-bearing, this is an alternativemodality for obtaining the motif-bearing peptides correlated with theparticular HLA molecule expressed on the cell.

Alternatively, cell lines that do not express endogenous HLA moleculescan be transfected with an expression construct encoding a single HLAallele. These cells can then be used as described, i.e., they can thenbe transfected with nucleic acids that encode 193P1E1B to isolatepeptides corresponding to 193P1E1B that have been presented on the cellsurface. Peptides obtained from such an analysis will bear motif(s) thatcorrespond to binding to the single HLA allele that is expressed in thecell.

As appreciated by one in the art, one can perform a similar analysis ona cell bearing more than one HLA allele and subsequently determinepeptides specific for each HLA allele expressed. Moreover, one of skillwould also recognize that means other than transfection, such as loadingwith a protein antigen, can be used to provide a source of antigen tothe cell.

Example 34 Complementary Polynucleotides

Sequences complementary to the 193P1E1B-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring 193P1E1B. Although use of oligonucleotidescomprising from about 15 to 30 base pairs is described, essentially thesame procedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06software (National Biosciences) and the coding sequence of 193P1E1B. Toinhibit transcription, a complementary oligonucleotide is designed fromthe most unique 5′ sequence and used to prevent promoter binding to thecoding sequence. To inhibit translation, a complementary oligonucleotideis designed to prevent ribosomal binding to a 193P1E1B-encodingtranscript.

Example 35 Purification of Naturally-Occurring or Recombinant 193P1E1BUsing 193P1E1B-Specific Antibodies

Naturally occurring or recombinant 193P1E1B is substantially purified byimmunoaffinity chromatography using antibodies specific for 193P1E1B. Animmunoaffinity column is constructed by covalently couplinganti-193P1E1B antibody to an activated chromatographic resin, such asCNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After thecoupling, the resin is blocked and washed according to themanufacturer's instructions.

Media containing 193P1E1B are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of 193P1E1B (e.g., high ionic strength buffers in thepresence of detergent). The column is eluted under conditions thatdisrupt antibody/193P1E1B binding (e.g., a buffer of pH 2 to pH 3, or ahigh concentration of a chaotrope, such as urea or thiocyanate ion), andGCR.P is collected.

Example 36 Identification of Molecules Which Interact with 193P1E1B

193P1E1B, or biologically active fragments thereof, are labeled with 1211 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled 193P1E1B, washed, andany wells with labeled 193P1E1B complex are assayed. Data obtained usingdifferent concentrations of 193P1E1B are used to calculate values forthe number, affinity, and association of 193P1E1B with the candidatemolecules.

Example 37 In Vivo Assay for 193P1E1B Tumor Growth Promotion

The effect of a 193P1E1B protein on tumor cell growth can be confirmedin vivo by gene overexpression in a variety of cancer cells such asthose in Table I. For example, SCID mice can be injected SQ on eachflank with 1×10⁶ prostate, kidney, colon or bladder cancer cells (suchas PC3, LNCaP, SCaBER, UM-UC-3, HT1376, SK-CO, Caco, RT4, T24, Caki,A-498 and SW839 cells) containing tkNeo empty vector or 193P1E1B.

At least two strategies can be used:

(1) Constitutive 193P1E1B expression under regulation of a promoter suchas a constitutive promoter obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2, 211, 504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus40 (SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, provided such promoters arecompatible with the host cell systems.

(2) Regulated expression under control of an inducible vector system,such as ecdysone, tet, etc., can be used provided such promoters arecompatible with the host cell systems. Tumor volume is then monitored atthe appearance of palpable tumors or by following serum markers such asPSA. Tumor development is followed over time to validate that193P1E1B-expressing cells grow at a faster rate and/or that tumorsproduced by 193P1E1B-expressing cells demonstrate characteristics ofaltered aggressiveness (e.g., enhanced metastasis, vascularization,reduced responsiveness to chemotherapeutic drugs). Tumor volume isevaluated by caliper measurements. Additionally, mice can be implantedwith the same cells orthotopically in the prostate, bladder, colon orkidney to determine if 193P1E1B has an effect on local growth, e.g., inthe prostate, bladder, colon or kidney or on the ability of the cells tometastasize, specifically to lungs or lymph nodes (Saffran et al., ProcNatl Acad Sci USA. 2001, 98: 2658; Fu, X., et al., Int. J. Cancer, 1991.49: 938-939; Chang, S., et al., Anticancer Res., 1997, 17: 3239-3242;Peralta, E. A., et al., J. Urol., 1999. 162:1806-1811). For instance,the orthotopic growth of PC3 and PC3-193P1E1B can be compared in theprostate of SCID mice. Such experiments reveal the effect of 193P1E1B onorthotopic tumor growth, metastasis and/or angiogenic potential.

Furthermore, this assay is useful to confirm the inhibitory effect ofcandidate therapeutic compositions, such as 193P1E1B antibodies orintrabodies, and 193P1E1B antisense molecules or ribozymes, or 193P1E1Bdirected small molecules, on cells that express a 193P1E1B protein.

Example 38 193P1E1B Monoclonal Antibody-mediated Inhibition of ProstateTumors In Vivo.

The significant expression of 193P1E1B, in cancer tissues, together withits restricted expression in normal tissues makes 193P1E1B an excellenttarget for antibody therapy. Similarly, 193P1E1B is a target for Tcell-based immunotherapy. Thus, the therapeutic efficacy ofanti-193P1E1B mAbs is evaluated, e.g., in human prostate cancerxenograft mouse models using androgen-independent LAPC-4 and LAPC-9xenografts (Craft, N., et al. Cancer Res, 1999. 59(19): p. 5030-5036),kidney cancer xenografts (AGS-K3, AGS-K6), kidney cancer metastases tolymph node (AGS-K6 met) xenografts, and kidney cancer cell linestransfected with 193P1E1B, such as 769P-193P E1B, A498-193P1E1B.

Antibody efficacy on tumor growth and metastasis formation is studied,e.g., in mouse orthotopic prostate cancer xenograft models and mousekidney xenograft models. The antibodies can be unconjugated, asdiscussed in this example, or can be conjugated to a therapeuticmodality, as appreciated in the art. Anti-193P1E1B mAbs inhibitformation of both the androgen-dependent LAPC-9 and androgen-independentPC3-193P1E1B tumor xenografts. Anti-193P1E1B mAbs also retard the growthof established orthotopic tumors and prolonged survival of tumor-bearingmice. These results indicate the utility of anti-193P1E1B mAbs in thetreatment of local and advanced stages of, e.g., prostate cancer. (See,e.g., Saffran, D., et al., PNAS 10:1073-1078. Similarly, anti-193P1E1BmAbs inhibit formation of AGS-K3 and AGS-K6 tumors in SCID mice, andprevent or retard the growth A498-193P1E1B tumor xenografts. Theseresults indicate the use of anti-193P1E1B mAbs in the treatment ofprostate and/or kidney cancer.

Administration of the anti-193P1E1B mAbs leads to retardation ofestablished orthotopic tumor growth and inhibition of metastasis todistant sites, resulting in a significant prolongation in the survivalof tumor-bearing mice. These studies indicate that 193P1E1B is anattractive target for immunotherapy and demonstrate the therapeutic useof anti-193P1E1B mAbs for the treatment of local and metastatic cancer.This example demonstrates that unconjugated 193P1E1B monoclonalantibodies are effective to inhibit the growth of human prostate tumorxenografts and human kidney xenografts grown in SCID mice.

Tumor Inhibition Using Multiple Unconjugated 193P1E1B mAbs

Materials and Methods

193P1E1B Monoclonal Antibodies:

Monoclonal antibodies are obtained against 193P1E1B, as described inExample 11 entitled: Generation of 193P1E1B Monoclonal Antibodies(mAbs), or may be obtained commercially. The antibodies arecharacterized by ELISA, Western blot, FACS, and immunoprecipitation fortheir capacity to bind 193P1E1B. Epitope mapping data for theanti-193P1E1B mAbs, as determined by ELISA and Western analysis,recognize epitopeson a 193P1E1B protein. Immunohistochemical analysis ofcancer tissues and cells is performed with these antibodies.

The monoclonal antibodies are purified from ascites or hybridoma tissueculture supernatants by Protein-G Sepharose chromatography, dialyzedagainst PBS, filter sterilized, and stored at −20° C. Proteindeterminations are performed by a Bradford assay (Bio-Rad, Hercules,Calif.). A therapeutic monoclonal antibody or a cocktail comprising amixture of individual monoclonal antibodies is prepared and used for thetreatment of mice receiving subcutaneous or orthotopic injections of,e.g., LAPC-9 prostate tumor xenografts.

Cancer Xenografts and Cell Lines

The LAPC-9 xenograft, which expresses a wild-type androgen receptor andproduces prostate-specific antigen (PSA), is passaged in 6- to8-week-old male ICR-severe combined immunodeficient (SCID) mice (TaconicFarms) by subcutaneous (s.c.) trocar implant (Craft, N., et al., 1999,Cancer Res. 59:5030-5036). The AGS-K3 and AGS-K6 kidney xenografts arealso passaged by subcutaneous implants in 6- to 8-week old SCID mice.Single-cell suspensions of tumor cells are prepared as described inCraft, et al. The prostate carcinoma cell line PC3 (American TypeCulture Collection) is maintained in RPMI supplemented with L-glutamineand 10% FBS, and the kidney carcinoma line A498 (American Type CultureCollection) is maintained in DMEM supplemented with L-glutamine and 10%FBS.

PC3-193P1E1B and A498-193P1E1B cell populations are generated byretroviral gene transfer as described in Hubert, R. S., et al., STEAP: AProstate-specific Cell-surface Antigen Highly Expressed in HumanProstate Tumors, Proc Nat. Acad. Sci. USA, 1999. 96(25): p. 14523-14528.Anti-193P1E1B staining is detected by using, e.g., an FITC-conjugatedgoat anti-mouse antibody (Southern Biotechnology Associates) followed byanalysis on a Coulter Epics-XL flow cytometer.

Xenograft Mouse Models.

Subcutaneous (s.c.) tumors are generated by injection of 1×10⁶ LAPC-9,AGS-K3, AGS-K6, PC3, PC3-193P1E1B, A498 or A498-193P1E1B cells mixed ata 1:1 dilution with Matrigel (Collaborative Research) in the right flankof male SCID mice. To test antibody efficacy on tumor formation, i.p.antibody injections are started on the same day as tumor-cellinjections. As a control, mice are injected with either purified mouseIgG (ICN) or PBS; or a purified monoclonal antibody that recognizes anirrelevant antigen not expressed in human cells. In preliminary studies,no difference is found between mouse IgG or PBS on tumor growth. Tumorsizes are determined by vemier caliper measurements, and the tumorvolume is calculated as length×width×height. Mice with s.c. tumorsgreater than 1.5 cm in diameter are sacrificed. PSA levels aredetermined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).Circulating levels of anti-193P1E1B mAbs are determined by a captureELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran,D., et al., PNAS 10:1073-1078 .

Orthotopic prostate injections are performed under anesthesia by usingketamine/xylazine. For prostate orthotopic studies, an incision is madethrough the abdominal muscles to expose the bladder and seminalvesicles, which then are delivered through the incision to expose thedorsal prostate. LAPC-9 cells (5×10⁵) mixed with Matrigel are injectedinto each dorsal lobe in a 10 μl volume. To monitor tumor growth, miceare bled on a weekly basis for determination of PSA levels. For kidneyorthotopic models, an incision is made through the abdominal muscles toexpose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel areinjected under the kidney capsule. The mice are segregated into groupsfor appropriate treatments, with anti-193P1E1B or control mAbs beinginjected i.p.

Anti-193P1E1B mAbs Inhibit Growth of 193P1E1B-ExpressingXenograft-Cancer Tumors

The effect of anti-193P1E1B mAbs on tumor formation is tested by using,e.g., LAPC-9 and/or AGS-K3 orthotopic models. As compared with the s.c.tumor model, the orthotopic model, which requires injection of tumorcells directly in the mouse prostate or kidney, respectively, results ina local tumor growth, development of metastasis in distal sites,deterioration of mouse health, and subsequent death (Saffran, D., etal., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90;Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make theorthotopic model more representative of human disease progression andallow for tracking of the therapeutic effect of mAbs on clinicallyrelevant end points.

Accordingly, tumor cells are injected into the mouse prostate or kidney,and the mice are segregated into two groups and treated with either: a)200-500 μg of anti-193P1E1B Ab, or b) PBS for two to five weeks.

As noted, a major advantage of the orthotopic prostate-cancer model isthe ability to study the development of metastases. Formation ofmetastasis in mice bearing established orthotopic tumors is studied byIHC analysis on lung sections using an antibody against aprostate-specific cell-surface protein STEAP expressed at high levels inLAPC-9 xenografts (Hubert, R. S., et al., Proc Natl. Acad. Sci. USA,1999. 96(25): p. 14523-14528) or anti-G250 antibody for kidney cancermodels. G250 is a clinically relevant marker for renal clear cellcarcinoma, which is selectively expressed on tumor but not normal kidneycells (Grabmaier K et al., Int J Cancer. 2000, 85: 865).

Mice bearing established orthotopic LAPC-9 tumors are administered500-1000 μg injections of either anti-193P1E1B mAb or PBS over a 4-weekperiod. Mice in both groups are allowed to establish a high tumor burden(PSA levels greater than 300 μg/ml), to ensure a high frequency ofmetastasis formation in mouse lungs. Mice then are killed and theirprostate/kidney and lungs are analyzed for the presence of tumor cellsby IHC analysis. These studies demonstrate a broad anti-tumor efficacyof anti-193P1E1B antibodies on initiation and/or progression of prostateand kidney cancer in xenograft mouse models. Anti-193P1E1B antibodiesinhibit tumor formation of both androgen-dependent andandrogen-independent prostate tumors as well as retarding the growth ofalready established tumors and prolong the survival of treated mice.Moreover, anti-193P1E1B mAbs demonstrate a dramatic inhibitory effect onthe spread of local prostate tumor to distal sites, even in the presenceof a large tumor burden. Similar therapeutic effects are seen in thekidney cancer model. Thus, anti-193P1E1B mAbs are efficacious on majorclinically relevant end points (tumor growth), prolongation of survival,and health.

Example 39 Therapeutic and Diagnostic Use of Anti-193P1E1B Antibodies inHumans.

Anti-193P1E1B monoclonal antibodies are safely and effectively used fordiagnostic, prophylactic, prognostic and/or therapeutic purposes inhumans. Western blot and immunohistochemical analysis of cancer tissuesand cancer xenografts with anti-193P1E1B mAb show strong extensivestaining in carcinoma but significantly lower or undetectable levels innormal tissues. Detection of 193P1E1B in carcinoma and in metastaticdisease demonstrates the usefulness of the mAb as a diagnostic and/orprognostic indicator. Anti-193P1E1B antibodies are therefore used indiagnostic applications such as immunohistochemistry of kidney biopsyspecimens to detect cancer from suspect patients.

As determined by flow cytometry, anti-193P1E1B mAb specifically binds tocarcinoma cells. Thus, anti-193P1E1B antibodies are used in diagnosticwhole body imaging applications, such as radioimmunoscintigraphy andradioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res20(2A):925-948 (2000)) for the detection of localized and metastaticcancers that exhibit expression of 193P1E1B. Shedding or release of anextracellular domain of 193P1E1B into the extracellular milieu, such asthat seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology27:563-568 (1998)), allows diagnostic detection of 193P1E1B byanti-193P1E1B antibodies in serum and/or urine samples from suspectpatients.

Anti-193P1E1B antibodies that specifically bind 193P1E1B are used intherapeutic applications for the treatment of cancers that express193P1E1B. Anti-193P1E1B antibodies are used as an unconjugated modalityand as conjugated form in which the antibodies are attached to one ofvarious therapeutic or imaging modalities well known in the art, such asa prodrugs, enzymes or radioisotopes. In preclinical studies,unconjugated and conjugated anti-193P1E1B antibodies are tested forefficacy of tumor prevention and growth inhibition in the SCID mousecancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6,(see, e.g., the Example entitled “193P1E1B Monoclonal Antibody-mediatedInhibition of Bladder and Lung Tumors In Vivo”). Either conjugated andunconjugated anti-193P1E1B antibodies are used as a therapeutic modalityin human clinical trials either alone or in combination with othertreatments as described in following Examples.

Example 40 Human Clinical Trials for the Treatment and Diagnosis ofHuman Carcinomas Through use of Human Anti-193P1E1B Antibodies In Vivo

Antibodies are used in accordance with the present invention whichrecognize an epitope on 193P1E1B, and are used in the treatment ofcertain tumors such as those listed in Table I. Based upon a number offactors, including 193P1E1B expression levels, tumors such as thoselisted in Table I are presently preferred indications. In connectionwith each of these indications, three clinical approaches aresuccessfully pursued.

I.) Adjunctive therapy: In adjunctive therapy, patients are treated withanti-193P1E1B antibodies in combination with a chemotherapeutic orantineoplastic agent and/or radiation therapy. Primary cancer targets,such as those listed in Table I, are treated under standard protocols bythe addition anti-193P1E1B antibodies to standard first and second linetherapy. Protocol designs address effectiveness as assessed by reductionin tumor mass as well as the ability to reduce usual doses of standardchemotherapy. These dosage reductions allow additional and/or prolongedtherapy by reducing dose-related toxicity of the chemotherapeutic agent.Anti-193P1E1B antibodies are utilized in several adjunctive clinicaltrials in combination with the chemotherapeutic or antineoplastic agentsadriamycin (advanced prostrate carcinoma), cisplatin (advanced head andneck and lung carcinomas), taxol (breast cancer), and doxorubicin(preclinical).

II.) Monotherapy: In connection with the use of the anti-193P1E1Bantibodies in monotherapy of tumors, the antibodies are administered topatients without a chemotherapeutic or antineoplastic agent. In oneembodiment, monotherapy is conducted clinically in end stage cancerpatients with extensive metastatic disease. Patients show some diseasestabilization. Trials demonstrate an effect in refractory patients withcancerous tumors.

III.) Imaging Agent: Through binding a radionuclide (e.g., iodine oryttrium (I¹³¹, Y⁹⁰) to anti-193P1E1B antibodies, the radiolabeledantibodies are utilized as a diagnostic and/or imaging agent. In such arole, the labeled antibodies localize to both solid tumors, as well as,metastatic lesions of cells expressing 193P1E1B. In connection with theuse of the anti-193P1E1B antibodies as imaging agents, the antibodiesare used as an adjunct to surgical treatment of solid tumors, as both apre-surgical screen as well as a post-operative follow-up to determinewhat tumor remains and/or returns. In one embodiment, a (¹¹¹In)-193P1E1Bantibody is used as an imaging agent in a Phase I human clinical trialin patients having a carcinoma that expresses 193P1E1B (by analogy see,e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients arefollowed with standard anterior and posterior gamma camera. The resultsindicate that primary lesions and metastatic lesions are identified

Dose and Route of Administration

As appreciated by those of ordinary skill in the art, dosingconsiderations can be determined through comparison with the analogousproducts that are in the clinic. Thus, anti-193P1E1B antibodies can beadministered with doses in the range of 5 to 400 mg/m², with the lowerdoses used, e.g., in connection with safety studies. The affinity ofanti-193P1E1B antibodies relative to the affinity of a known antibodyfor its target is one parameter used by those of skill in the art fordetermining analogous dose regimens. Further, anti-193P1E1B antibodiesthat are fully human antibodies, as compared to the chimeric antibody,have slower clearance; accordingly, dosing in patients with such fullyhuman anti-193P1E1B antibodies can be lower, perhaps in the range of 50to 300 mg/m² and still remain efficacious. Dosing in mg/m², as opposedto the conventional measurement of dose in mg/kg, is a measurement basedon surface area and is a convenient dosing measurement that is designedto include patients of all sizes from infants to adults.

Three distinct delivery approaches are useful for delivery ofanti-193P1E1B antibodies. Conventional intravenous delivery is onestandard delivery technique for many tumors. However, in connection withtumors in the peritoneal cavity, such as tumors of the ovaries, biliaryduct, other ducts, and the like, intraperitoneal administration mayprove favorable for obtaining high dose of antibody at the tumor and toalso minimize antibody clearance. In a similar manner, certain solidtumors possess vasculature that is appropriate for regional perfusion.Regional perfusion allows for a high dose of antibody at the site of atumor and minimizes short term clearance of the antibody.

Clinical Development Plan (CDP)

Overview: The CDP follows and develops treatments of anti-193P1E1Bantibodies in connection with adjunctive therapy, monotherapy, and as animaging agent. Trials initially demonstrate safety and thereafterconfirm efficacy in repeat doses. Trails are open label comparingstandard chemotherapy with standard therapy plus anti-193P1E1Bantibodies. As will be appreciated, one criteria that can be utilized inconnection with enrollment of patients is 193P1E1B expression levels intheir tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic, safetyconcerns are related primarily to (i) cytokine release syndrome, i.e.,hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 193P1E1B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Anti-193P1E1B antibodies are found to be safe uponhuman administration.

Example 41 Human Clinical Trial Adjunctive Therapy with HumanAnti-193P1E1B Antibody and Chemotherapeutic Agent

A phase I human clinical trial is initiated to assess the safety of sixintravenous doses of a human anti-193P1E1B antibody in connection withthe treatment of a solid tumor, e.g., a cancer of a tissue listed inTable I. In the study, the safety of single doses of anti-193P1E1Bantibodies when utilized as an adjunctive therapy to an antineoplasticor chemotherapeutic agent as defined herein, such as, withoutlimitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or thelike, is assessed. The trial design includes delivery of six singledoses of an anti-193P1E1B antibody with dosage of antibody escalatingfrom approximately about 25 mg/m²to about 275 mg/m²over the course ofthe treatment in accordance with the following schedule:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 mAb Dose 25 75 125 175 225 275mg/m² mg/m² mg/m² mg/m² mg/m² mg/m² Chemotherapy + + + + + + (standarddose)

Patients are closely followed for one-week following each administrationof antibody and chemotherapy. In particular, patients are assessed forthe safety concerns mentioned above: (i).cytokine release syndrome,i.e., hypotension, fever, shaking, chills; (ii) the development of animmunogenic response to the material (i.e., development of humanantibodies by the patient to the human antibody therapeutic, or HAHAresponse); and, (iii) toxicity to normal cells that express 193P1E1B.Standard tests and follow-up are utilized to monitor each of thesesafety concerns. Patients are also assessed for clinical outcome, andparticularly reduction in tumor mass as evidenced by MRI or otherimaging.

The anti-193P1E1B antibodies are demonstrated to be safe andefficacious, Phase II trials confirm the efficacy and refine optimumdosing.

Example 42 Human Clinical Trial: Monotherapy with Human Anti-193P1E1BAntibody

Anti-193P1E1B antibodies are safe in connection with the above-discussedadjunctive trial, a Phase II human clinical trial confirms the efficacyand optimum dosing for monotherapy. Such trial is accomplished, andentails the same safety and outcome analyses, to the above-describedadjunctive trial with the exception being that patients do not receivechemotherapy concurrently with the receipt of doses of anti-193P1E1Bantibodies.

Example 43 Human Clinical Trial: Diagnostic Imaging with Anti-193P1E1BAntibody

Once again, as the adjunctive therapy discussed above is safe within thesafety criteria discussed above, a human clinical trial is conductedconcerning the use of anti-193P1E1B antibodies as a diagnostic imagingagent. The protocol is designed in a substantially similar manner tothose described in the art, such as in Divgi et al. J. Natl. CancerInst. 83:97-104 (1991). The antibodies are found to be both safe andefficacious when used as a diagnostic modality.

Example 44 Homology Comparison of 193P1E1B to Known Sequences

The 193P1E1B protein has several forms, including 3 SNPs and 5 splicevariants (FIG. 4G). Three variants, namely 193P1E1B v. 1, v.5 and v.6,consist of 412 amino acids each, with calculated molecular weight of46.25 kDa, and pl of 5.18, and differ from each other by one amino acid.193P1E1B v.10, v.9 and v.12 are progressively smaller proteins, with388, 330, and 73 amino acids respectively. These variants differ withregards to their molecular weights and isoelectic points, as shown inTable L. All variants of 193P1E1B are predicted to be nuclear proteins,with possible localization to the mitochondria (193P1E3B v.1, v.5, v.6,v.9, v.10, v.11 and v.13) or cytoplasm (193P1E3B v.12). Motif analysisrevealed no known motifs.

All protein variants of 193P1E1B show best homology to a human un-namedprotein (gi 21748775) of unknown function, with 193P1E1B v.5 showing100% identity with gi 21748775 over the entire length of the protein,and 193P1E1B sharing 99% identity with the same protein. Similarly, theother variants show highest homology to the human un-named protein (gi21748775). The variant with the lowest homology to gi 21748775 is193P1E1B v.12, with 89% identity and 89% homology over the first 39amino acids of the protein (FIGS. 4A-D).

The 193P1E1B protein shows homology to a protein of known function,namely the arginine repressor (gi14349114) of E. coli, also known ascarbamate kinase. Variant 193P1E1B v.1 shows 30% identity and 57%homology with that protein (FIG. 4E). This homology indicates that193P1E1B may regulate ATP synthesis and metabolism (Marina A et al., EurJ Biochem 1998, 253:280; Alcantara C et al., FEBS Left. 2000, 484:261),a key factor in cell growth and biological function.

In addition, 193P1E1B also exhibit some homology to humandouble-stranded RNA-specific adenosine deaminase (ADAR-c isoform) (gi7669475). 193P1E1Bv.1 shares 26% identity and 40% homology with ADAR-c(FIG. 4F). Similar results were obtained with 193P1E1Bv.5, v.6, v.9,v.10 and v.13. This suggests that 193P1E1B has the ability to bindspecifically to double stranded RNA or DNA (Schwartz T., et al., NatureStruc. Biol. 2001, 8:761). Adenosine deaminases acting on RNA have beenshown to be involved in RNA editing (Raitskin, O., et al., Proc. Natl.Acad. Sci 2001, 98:6571). Recent studies have associated adenosinedeaminase with cancer and cellular proliferation (Eroglu A, et al., MedOncol. 2000, 17:319-24; Barry C. P., and, Lind, S. E., Cancer Res. 2000,60:1887-94). In addition, adenosine deaminase is highly expressed intumor tissue relative to normal tissues in such cancers as colon,leukemia and other lymphoid cancers (Blatt, J., et al., N Engl J Med.1980; 303:918; Eroglu, A., et al., Med Oncol. 2000, 17:319). Adenosinedeaminase has been considered a potential marker for lymphoidmalignancies (Blatt J et al., N Engl J Med. 1980;303: 918). In addition,inhibition of adenosine deaminase was found to result in cell death ofepithelial cells (Barry, C. P., and, Lind, S. E., Cancer Res. 2000,60:1887).

This information indicates that 193P1E1B plays a role in thetransformation of mammalian cells, supports cell survival andproliferation, and regulates gene transcription by regulating events inthe nucleus. Accordingly, when 193P1E1B functions as a regulator of celltransformation, tumor formation, or as a modulator of transcriptioninvolved in activating genes associated with inflammation, tumorigenesisor proliferation, 193P1E1B is used for therapeutic, diagnostic,prognostic and/or preventative purposes.

Example 45 Identification and Confirmation of Potential SignalTransduction Pathways

Many mammalian proteins have been reported to interact with signalingmolecules and to participate in regulating signaling pathways. (JNeurochem. 2001; 76:217-223). In particular, adenosine deaminase hasbeen found to associate with G-proteins, thereby regulating severalsignaling pathways (Ciruela F et al., FEBS Lett. 1996, 380:219). Usingimmunoprecipitation and Western blotting techniques, proteins areidentified that associate with 193P1E1B and mediate signaling events.Several pathways known to play a role in cancer biology can be regulatedby 193P1E1B, including phospholipid pathways such as P13K, AKT, etc,adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as wellas mitogenic/survival cascades such as ERK, p38, etc .(Cell GrowthDiffer. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000,19:3003, J. Cell Biol. 1997, 138:913).

To confirm that 193P1E1B directly or indirectly activates known signaltransduction pathways in cells, luciferase (luc) based transcriptionalreporter assays are carried out in cells expressing individual genes.These transcriptional reporters contain consensus-binding sites forknown transcription factors that lie downstream of well-characterizedsignal transduction pathways. The reporters and examples of theseassociated transcription factors, signal transduction pathways, andactivation stimuli are listed below.

-   -   1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress    -   2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation    -   3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress    -   4. ARE-luc, androgen receptor; steroids/MAPK;        growth/differentiation/apoptosis    -   5. p53-luc, p53; SAPK; growth/differentiation/apoptosis    -   6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress

Gene-mediated effects can be assayed in cells showing mRNA expression.Luciferase reporter plasmids can be introduced by lipid-mediatedtransfection (TFX-50, Promega). Luciferase activity, an indicator ofrelative transcriptional activity, is measured by incubation of cellextracts with luciferin substrate and luminescence of the reaction ismonitored in a luminometer.

Signaling pathways activated by 193P1E1B are mapped and used for theidentification and validation of therapeutic targets. When 193P1E1B isinvolved in cell signaling, it is used as target for diagnostic,prognostic, preventative and/or therapeutic purposes.

Example 46 Regulation of Transcription

The nuclear localization of 193P1E1B and its ability to regulateadenosine deaminase indicate that it is effectively used as a modulatorof the transcriptional regulation of eukaryotic genes. Regulation ofgene expression is confirmed, e.g., by studying gene expression in cellsexpressing or lacking 193P1E1B. For this purpose, two types ofexperiments are performed.

In the first set of experiments, RNA from parental and193P1E1B-expressing cells are extracted and hybridized to commerciallyavailable gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer.2000. 83:246). Resting cells as well as cells treated with FBS,pheromones, or growth factors are compared. Differentally expressedgenes are identified in accordance with procedures known in the art. Thedifferentially expressed genes are then mapped to biological pathways(Chen K et al. Thyroid. 2001. 11:41.).

In the second set of experiments, specific transcriptional pathwayactivation is evaluated using commercially available (Stratagene)luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc,ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters containconsensus binding sites for known transcription factors that liedownstream of well-characterized signal transduction pathways, andrepresent a good tool to ascertain pathway activation and screen forpositive and negative modulators of pathway activation. Thus, 193P1E1Bplays a role in gene regulation, and it is used as a target fordiagnostic, prognostic, preventative and/or therapeutic purposes.

Example 47 Involvement in Tumor Progression

The 193P1E1B gene can contribute to the growth of cancer cells. The roleof 193P1E1B in tumor growth is confirmed in a variety of primary andtransfected cell lines including prostate, colon, bladder and kidneycell lines, as well as NIH 3T3 cells engineered to stably express193P1E1B. Parental cells lacking 193P1E1B and cells expressing 193P1E1Bare evaluated for cell growth using a well-documented proliferationassay (Fraser S P, et al., Prostate 2000;44:61, Johnson DE, Ochieng J,Evans SL. Anticancer Drugs. 1996, 7:288). The effect of 193P1E1B canalso be observed on cell cycle progression. Control and193P1E1B-expressing cells are grown in low serum overnight, and treatedwith 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdU andpropidium iodide incorporation by FACS analysis.

To confirm the role of 193P1E1B in the transformation process, itseffect in colony forming assays is investigated. Parental NIH-3T3 cellslacking 193P1E1B are compared to NIH-3T3 cells expressing 193P1E1B,using a soft agar assay under stringent and more permissive conditions(Song Z. et al. Cancer Res. 2000;60:6730).

To confirm the role of 193P1E1B in invasion and metastasis of cancercells, a well-established assay is used. A non-limiting example is theuse of an assay which provides a basement membrane or an analog thereofused to detect whether cells are invasive (e.g., a Transwell InsertSystem assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Controlcells, including prostate, and bladder cell lines lacking 193P1E1B arecompared to cells expressing 193P1E1B. Cells are loaded with thefluorescent dye, calcein, and plated in the top well of a supportstructure coated with a basement membrane analog (e.g. the Transwellinsert) and used in the assay. Invasion is determined by fluorescence ofcells in the lower chamber relative to the fluorescence of the entirecell population.

193P1E1B can also play a role in cell cycle and apoptosis. Parentalcells and cells expressing 193P1E1B are compared for differences in cellcycle regulation using a well-established BrdU assay (Abdel-Malek ZA. JCell Physiol. 1988, 136:247). In short, cells are grown under bothoptimal (full serum) and limiting (low serum) conditions are labeledwith BrdU and stained with anti-BrdU Ab and propidium iodide. Cells areanalyzed for entry into the G1, S, and G2M phases of the cell cycle.Alternatively, the effect of stress on apoptosis is evaluated in controlparental cells and cells expressing 193P1E1B, including normal and tumorprostate, and kidney cells. Engineered and parental cells are treatedwith various chemotherapeutic agents, such as etoposide, flutamide, etc,and protein synthesis inhibitors, such as cycloheximide. Cells arestained with annexin V-FITC and cell death is measured by FACS analysis.The modulation of cell death by 193P1E1B can play a critical role inregulating tumor progression and tumor load.

When 193P1E1B plays a role in cell growth, transformation, invasion orapoptosis, it is used as a target for diagnostic, prognostic,preventative and/or therapeutic purposes.

Example 48 Involvement in Angiogenesis

Angiogenesis or new capillary blood vessel formation is necessary fortumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J.Endocrinology. 1998 139:441). Based on the effect of phsophodieseteraseinhibitors on endothelial cells, 193P1E1B plays a role in angiogenesis(DeFouw L et al., Microvasc Res 2001, 62:263). Several assays have beendeveloped to measure angiogenesis in vitro and in vivo, such as thetissue culture assays endothelial cell tube formation and endothelialcell proliferation. Using these assays as well as in vitroneo-vascularization, the role of 193P1E1B in angiogenesis, enhancementor inhibition, is confirmed. For example, endothelial cells engineeredto express 193P1E1B are evaluated using tube formation and proliferationassays. The effect of 193P1E1B is also confirmed in animal models invivo. For example, cells either expressing or lacking 193P1E1B areimplanted subcutaneously in immunocompromised mice. Endothelial cellmigration and angiogenesis are evaluated 5-15 days later usingimmunohistochemistry techniques. 193P1E1B affects angiogenesis, and itis used as a target for diagnostic, prognostic, preventative and/ortherapeutic purposes.

Example 49 Involvement in Cell Adhesion

Cell adhesion plays a critical role in tissue colonization andmetastasis. 193P1E1B can participate in cellular organization, and as aconsequence cell adhesion and motility. To confirm that 193P1E1Bregulates cell adhesion, control cells lacking 193P1E1B are compared tocells expressing 193P1E1B, using techniques previously described (see,e.g., Haier et al., Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J.Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cellslabeled with a fluorescent indicator, such as calcein, are incubated ontissue culture wells coated with media alone or with matrix proteins.Adherent cells are detected by fluorimetric analysis and percentadhesion is calculated. In another embodiment, cells lacking orexpressing 193P1E1B are analyzed for their ability to mediate cell-celladhesion using similar experimental techniques as described above. Bothof these experimental systems are used to identify proteins, antibodiesand/or small molecules that modulate cell adhesion to extracellularmatrix and cell-cell interaction. Cell adhesion plays a critical role intumor growth, progression, and, colonization, and 193P1E1B is involvedin these processes. Thus, it serves as a diagnostic, prognostic,preventative and/or therapeutic modality.

Example 50 Protein-Protein Association

Several adenosine deaminasess have been shown to interact with otherproteins, thereby regulating gene transcription, protein function, aswell as cell growth (Raitskin et al above; Morimoto C, and SchlossmanSF, Immunol Rev. 1998, 161:55.). Using immunoprecipitation techniques aswell as two yeast hybrid systems, proteins are identified that associatewith 193P1E1B. Immunoprecipitates from cells expressing 193P1E1B andcells lacking 193P1E1B are compared for specific protein-proteinassociations.

Studies are performed to confirm the extent of association of 193P1E1Bwith effector molecules, such as nuclear proteins, transcriptionfactors, kinases, phosphates, etc. Studies comparing 193P1E1B positiveand 193P1E1B negative cells as well as studies comparingunstimulated/resting cells and cells treated with epithelial cellactivators, such as cytokines, growth factors, androgen andanti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are confirmed using two yeasthybrid methodology (Curr. Opin. Chem. Biol. 1999, 3:64). A vectorcarrying a library of proteins fused to the activation domain of atranscription factor is introduced into yeast expressing a193P1E1B-DNA-binding domain fusion protein and a reporter construct.Protein-protein interaction is detected by colorimetric reporteractivity. Specific association with effector molecules and transcriptionfactors directs one of skill to the mode of action of 193P1E1B, and thusidentifies therapeutic, prognostic, preventative and/or diagnostictargets for cancer. This and similar assays are also used to identifyand screen for small molecules that interact with 193P1E1B. Thus it isfound that 193P1E1B associates with proteins and small molecules.Accordingly, 193P1E1Band these proteins and small molecules are used fordiagnostic, prognostic, preventative and/or therapeutic purposes.

Throughout this application, various website data content, publications,patent applications and patents are referenced. (Websites are referencedby their Uniform Resource Locator, or URL, addresses on the World WideWeb.) The disclosures of each of these references are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

Tables:

TABLE I Tissues that Express 193P1E1B: a. Malignant Tissues ProstateBladder Kidney Colon Lung Ovary Breast Pancreas Testis Uterus Skin Bone

TABLE II Amino Acid Abbreviations SINGLE LETTER THREE LETTER FULL NAME FPhe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine C Cyscysteine W Trp tryptophan P Pro proline H His histidine Q Gln glutamineR Arg arginine I Ile isoleucine M Met methionine T Thr threonine N Asnasparagine K Lys lysine V Val valine A Ala alanine D Asp aspartic acid EGlu glutamic acid G Gly glycine

Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitutionmatrix (block substitution matrix). The higher the value, the morelikely a substitution is found in related, natural proteins. A C D E F GH I K L M N P Q R S T V W Y . 4 0 −2 −1 −2 0 −2 −1 −1 −1 −1 −2 −1 −1 −11 0 0 −3 −2 A 9 −3 −4 −2 −3 −3 −1 −3 −1 −1 −3 −3 −3 −3 −1 −1 −1 −2 −2 C6 2 −3 −1 −1 −3 −1 −4 −3 1 −1 0 −2 0 −1 −3 −4 −3 D 5 −3 −2 0 −3 1 −3 −20 −1 2 0 0 −1 −2 −3 −2 E 6 −3 −1 0 −3 0 0 −3 −4 −3 −3 −2 −2 −1 1 3 F 6−2 −4 −2 −4 −3 0 −2 −2 −2 0 −2 −3 −2 −3 G 8 −3 −1 −3 −2 1 −2 0 0 −1 −2−3 −2 2 H 4 −3 2 1 −3 −3 −3 −3 −2 −1 3 −3 −1 I 5 −2 −1 0 −1 1 2 0 −1 −2−3 −2 K 4 2 −3 −3 −2 −2 −2 −1 1 −2 −1 L 5 −2 −2 0 −1 −1 −1 1 −1 −1 M 6−2 0 0 1 0 −3 −4 −2 N 7 −1 −2 −1 −1 −2 −4 −3 P 5 1 0 −1 −2 −2 −1 Q 5 −1−1 −3 −3 −2 R 4 1 −2 −3 −2 S 5 0 −2 −2 T 4 −3 −1 V 11 2 W 7 Y

TABLE IV (A): HLA Class I Supermotifs/Motifs POSITION POSITION POSITION3 (Primary C Terminus 2 (Primary Anchor) Anchor) (Primary Anchor)SUPERMOTIF A1 TI LVMS FWY (SEQ ID NO:124) A2 LIVM ATQ IV MATL (SEQ IDNO:125) (SEQ ID NO:126) A3 VSMA TLI RK (SEQ ID NO:127) A24 YF WIVLMT FIYWLM (SEQ ID NO:128) (SEQ ID NO:129) B7 P VILF MWYA (SEQ ID NO:130) B27RHK FYL WMIVA (SEQ ID NO:131) B44 E D FWYLIMVA (SEQ ID NO:132) B58 ATSFWY LIVMA (SEQ ID NO:133) B62 QL IVMP FWY MIVLA (SEQ ID NO:134) (SEQ IDNO:135) MOTIFS A1 TSM Y A1 DE AS Y (SEQ ID NO:247) A2.1 LM VQIAT V LIMAT(SEQ ID NO:136) (SEQ ID NO:137) A3 LMVISATF CGD KYR HFA (SEQ ID NO:138)(SEQ ID NO:139) A11 VTMLISAGN CDF K RYH (SEQ ID NO:140) (SEQ ID NO:141)A24 YFW M FLIW (SEQ ID NO:142) (SEQ ID NO:143) A*3101 MVT ALIS R K (SEQID NO:144) A*3301 MVALF IST RK (SEQ ID NO:145) A*6801 AVT MSLI RK (SEQID NO:146) B*0702 P LMF WYAIV (SEQ ID NO:147) B*3501 P LMFWY IVA (SEQ IDNO:148) B51 P LIVF WYAM (SEQ ID NO:149) B*5301 P IMFWY ALV (SEQ IDNO:150) B*5401 P ATIVL MFWY (SEQ ID NO:151) Bolded residues arepreferred, italicized residues are less preferred: A peptide isconsidered motif-bearing if it has primary anchors at each primaryanchor position for a motif or supermotif as specified in the abovetable.

TABLE IV (B): HLA Class II Supermotif 1 6 9 W, F, Y, V, I, L A, V, I, L,P, C, S, T A, V, I, L, C, S, T, M, Y

TABLE IV (C): HLA Class II Motifs MOTIFS 1° anchor 1 2 3 4 5 1° anchor 67 8 9 DR4 preferred FMYLIVW M T I VSTCPALIM MH MH deleterious (SEQ IDNO:152) W (SEQ ID NO:153) R WDE DR1 preferred MFLIVWY PAMQ VMATSPLIC MAVM deleterious (SEQ ID NO:154) C (SEQ ID NO:155) CWD (SEQ ID NO:156)GDE D CH FD DR7 preferred MFLIVWY M W A IVMSACTPL M IV deleterious (SEQID NO:157) C G (SEQ ID NO:158) GRD N G DR3 MOTIFS 1° anchor 1 2 3 1°anchor 4 5 1° anchor 6 Motif a preferred LIVMFY D (SEQ ID NO:159) Motifb preferred LIVMFAY DNQEST KRH (SEQ ID NO:160) (SEQ ID NO:161) DRSupermotif MFLIVWY VMSTACPLI (SEQ ID NO:162) (SEQ ID NO:163) Italicizedresidues indicate less preferred or “tolerated” residues

TABLE IV (D): HLA Class I Supermotifs SUPER- MOTIFS POSITION 1 2 3 4 5 67 8 C-terminus A1 1° Anchor 1° Anchor TILVMS FWY (SEQ ID NO:164) A2 1°Anchor 1° Anchor LIVMATQ LIVMAT (SEQ ID NO:165) (SEQ ID NO:166) A3Preferred 1° Anchor YFW YFW YFW P 1° Anchor VSMATLI (4/5) (3/5) (4/5)(4/5) RK (SEQ ID NO:167) deleterious DE (3/5); DE P (5/5) (4/5) A24 1°Anchor 1° Anchor YFWIVLMT FIYWLM (SEQ ID NO:168) (SEQ ID NO:169) B7Preferred FWY (5/5) 1° Anchor FWY FWY 1° Anchor LIVM (3/5) P (4/5) (3/5)VILFMWYA (SEQ ID NO:170) (SEQ ID NO:171) deleterious DE (3/5); P(5/5);DE G QN DE G(4/5); A(3/5); QN(3/5) (3/5) (4/5) (4/5) (4/5) B27 1° Anchor1° Anchor RHK FYLWMIVA (SEQ ID NO:172) B44 1° Anchor 1° Anchor EDFWYLIMVA (SEQ ID NO:173) B58 1° Anchor 1° Anchor ATS FWYLIVMA (SEQ IDNO:174) B62 1° Anchor 1° Anchor QLIVMP FWYMIVLA (SEQ ID NO:175) (SEQ IDNO:176) Italicized residues indicate less preferred or “tolerated”residues

TABLE IV (E): HLA Class I Motifs PO- SI- 9 or C- C- TION 1 2 3 4 5 6 7 8terminus terminus A1 pre- GFYW 1° Anchor DEA YFW P DEQN YFW 1° Anchor9-mer ferred (SEQ ID STM (SEQ ID Y NO:177) NO:178) del- DE RHKLIVMP A GA ete- (SEQ ID rious NO:179) A1 pre- GRHK ASTC- 1° Anchor GSTC ASTC (SEQLIVM (SEQ DE 1° Anchor 9-mer ferred (SEQ ID LIVM * DEAS (SEQ (SEQ ID IDNO:184) ID NO:185) Y NO:180) (SEQ ID ID NO:182) NO:183) NO:181) del- ARHKD- DE PQN RHK PG GP ete- EPYFW rious (SEQ ID NO:186) A1 pre- YFW 1°Anchor DEAQN A YFWQN PASTC GDE P 1° Anchor 10- ferred STM (SEQ ID (SEQID (SEQ ID Y mer NO:187) NO:188) NO:189) del- GP RHKGLIVM DE RHK QNA *RHKYFW RHK A ete- (SEQ ID (SEQ ID rious NO:190) NO:191) A1 pre- YFWSTCLIVM 1° Anchor A YFW PG G YFW 1° Anchor 10- ferred (SEQ ID DEAS (SEQY mer NO:192) ID NO:193) del- RHK RHKD- P G PRHK (SEQ QN ete- EPYFW IDNO:195) rious (SEQ ID NO:194) A2.1 pre- YFW 1° Anchor YFW STC YFW A P 1°Anchor 9-mer ferred LMIV- VLIMAT QAT (SEQ ID (SEQ ID NO:197) NO:196)del- DEP DERKH RKH DERKH ete- (SEQ ID (SEQ ID rious NO:198) NO:199) PO-SI- C- TION: 1 2 3 4 5 6 7 8 9 Terminus A2.1 pre- AYFW 1° Anchor LVIM(SEQ G G FYWLVIM 1° Anchor 10- ferred LMIV- ID NO:201) (SEQ ID VLIMATmer QAT NO:202) (SEQ ID (SEQ ID NO:203) NO:200) del- DEP DE RKHA P RKHDERKH RKH ete- (SEQ ID (SEQ ID rious NO:204) NO:205) A3 pre- RHK 1°Anchor YFW PRHKYFW A YFW P 1° Anchor ferred LMVIS- (SEQ ID KYRHFA ATFCGDNO:207) (SEQ ID (SEQ ID NO:208) NO:206) del- DEP DE ete- rious A11 pre-A 1° Anchor YFW YFW A YFW YFW P 1° Anchor ferred VTLMIS- KRYH AGNCDF(SEQ ID (SEQ ID NO:210) NO:209) del- DEP A G ete- rious A24 pre- YFWRHK1° Anchor STC YFW YFW 1° Anchor 9-mer ferred (SEQ ID YFWM FLIW NO:211)(SEQ ID (SEQ ID NO:212) NO:213) del- DEG DE G QNP DERHK G AQN ete- (SEQID rious NO:214) A24 Pre- 1° Anchor P YFWP P 1° Anchor 10- ferred YFWM(SEQ ID FLIW mer (SEQ ID NO:216) (SEQ ID NO:215) NO:217) Del- GDE QN RHKDE A QN DEA ete- rious A31- Pre- RHK 1° Anchor YFW P YFW YFW AP 1°Anchor 01 ferred MVTA- RK LIS (SEQ ID NO:218) Del- DEP DE ADE DE DE DEete- rious A33- Pre- 1° Anchor YFW AYFW 1° Anchor 01 ferred MVAL- (SEQID RK FIST NO:220) (SEQ ID NO:219) Del- GP DE ete- rious A68- Pre-YFWSTC 1° Anchor YFWLIVM YFW P 1° Anchor 01 ferred (SEQ ID AVTM- (SEQ IDRK NO:221) SLI NO:223) (SEQ ID NO:222) del- GP DEG RHK A ete- rious B07-Pre- RHKFWY 1° Anchor RHK RHK RHK RHK PA 1° Anchor 02 ferred (SEQ ID PLMFW- NO:224) YAIV (SEQ ID NO:225) del- DEQNP DEP DE DE GDE QN DE ete-(SEQ ID rious NO:226) B35- Pre- FWYL- 1° Anchor FWY FWY 1° Anchor 01ferred IVM P LMFW- (SEQ ID YIVA NO:227) (SEQ ID NO:228) del- AGP G Gete- rious B51 Pre- LIVM- 1° Anchor FWY STC FWY G FWY 1° Anchor ferredFWY P LIVF- (SEQ ID WYAM NO:229) (SEQ ID NO:230) del- AGPD- DE G DEQNGDE ete- ERHK (SEQ ID rious STC NO:232) (SEQ ID NO:231) B53- pre- LIVM-1° Anchor FWY STC FWY LIVMFWY FWY 1° Anchor 01 ferred FWY P (SEQ IDIMFW- (SEQ ID NO:234) YALV NO:233) (SEQ ID NO:235) del- AGPQN G RHKQN DEete- (SEQ ID (SEQ ID rious NO:236) NO:237) B54- pre- FWY 1° AnchorFWYLIVM LIVM ALIVM EWYAP 1° Anchor 01 ferred P (SEQ ID (SEQ ID (SEQ ID(SEQ ID ATIVL- NO:238) NO:239) NO:240) NO:241) MFWY (SEQ ID NO:242) del-GPQNDE GDESTC RHKDE DE QNDGE DE ete- (SEQ ID (SEQ ID (SEQ ID (SEQ IDrious NO:243) NO:244) NO:245) NO:246)

TABLE IV (F): Summary of HLA-supertypes Overall phenotypic frequenciesof HLA-supertypes in different ethnic populations Phenotypic frequencySpecificity Ave- Supertype Position 2 C-Terminus Caucasian N.A. BlackJapanese Chinese Hispanic rage B7 P AILMVFWY (SEQ ID NO:248) 43.2 55.157.1 43.0 49.3 49.5 A3 AILMVST (SEQ ID NO:249) RK 37.5 42.1 45.8 52.743.1 44.2 A2 AILMVT (SEQ ID NO:250) AILMVT (SEQ ID NO:251) 45.8 39.042.4 45.9 43.0 42.2 A24 YF (WIVLMT) (SEQ ID NO:252) FI (YWLM) (SEQ IDNO:253) 23.9 38.9 58.6 40.1 38.3 40.0 B44 E (D) FWYLIMVA (SEQ ID NO:254)43.0 21.2 42.9 39.1 39.0 37.0 A1 TI (LVMS) (SEQ ID NO:255) FWY 47.1 16.121.8 14.7 26.3 25.2 B27 RHK FYL (WMI) (SEQ ID NO:256) 28.4 26.1 13.313.9 35.3 23.4 B62 QL (IVMP) (SEQ ID NO:257) FWY (MIV) (SEQ ID NO:258)12.6 4.8 36.5 25.4 11.1 18.1 B58 ATS FWY (LIV) (SEQ ID NO:259) 10.0 25.11.6 9.0 5.9 10.3

TABLE IV (G): Calculated population coverage afforded by differentHLA-supertype combinations Phenotypic frequency HLA-supertypes CaucasianN.A Blacks Japanese Chinese Hispanic Average A2, A3 and B7 83.0 86.187.5 88.4 86.3 86.2 A2, A3, B7, A24, 99.5 98.1 100.0 99.5 99.4 99.3 B44and A1 99.9 99.6 100.0 99.8 99.9 99.8 A2, A3, B7, A24, B44, A1, B27,B62, and B58 Motifs indicate the residues defining supertypespecificites. The motifs incorporate residues determined on the basis ofpublished data to be recognized by multiple alleles within thesupertype. Residues within brackets are additional residues alsopredicted to be tolerated by multiple alleles within the supertype.

TABLE V Frequently Occurring Motifs avrg. % Name identity DescriptionPotential Function zf-C2H2 34% Zinc finger, C2H2 type Nucleicacid-binding protein functions as transcription factor, nuclear locationprobable cytochrome_b_N 68% Cytochrome b(N- membrane bound oxidase,generate terminal)/b6/petB superoxide lg 19% Immunoglobulin domaindomains are one hundred amino acids long and include a conservedintradomain disulfide bond. WD40 18% WD domain, G-beta repeat tandemrepeats of about 40 residues, each containing a Trp-Asp motif. Functionin signal transduction and protein interaction PDZ 23% PDZ domain mayfunction in targeting signaling molecules to sub-membranous sites LRR28% Leucine Rich Repeat short sequence motifs involved inprotein-protein interactions Pkinase 23% Protein kinase domain conservedcatalytic core common to both serine/threonine and tyrosine proteinkinases containing an ATP binding site and a catalytic site PH 16% PHdomain pleckstrin homology involved in intracellular signaling or asconstituents of the cytoskeleton EGF 34% EGF-like domain 30-40amino-acid long found in the extracellular domain of membrane- boundproteins or in secreted proteins Rvt 49% Reverse transcriptase(RNA-dependent DNA polymerase) Ank 25% Ank repeat Cytoplasmic protein,associates integral membrane proteins to the cytoskeleton Oxidored_q132% NADH- membrane associated. Involved in Ubiquinone/plastoquinoneproton translocation across the (complex I), various chains membraneEfhand 24% EF hand calcium-binding domain, consists of a12 residue loopflanked on both sides by a 12 residue alpha-helical domain Rvp 79%Retroviral aspartyl Aspartyl or acid proteases, centered on protease acatalytic aspartyl residue Collagen 42% Collagen triple helix repeatextracellular structural proteins involved (20 copies) in formation ofconnective tissue. The sequence consists of the G-X-Y and thepolypeptide chains forms a triple helix. Fn3 20% Fibronectin type IIIdomain Located in the extracellular ligand- binding region of receptorsand is about 200 amino acid residues long with two pairs of cysteinesinvolved in disulfide bonds 7tm_1 19% 7 transmembrane receptor sevenhydrophobic transmembrane (rhodopsin family) regions, with theN-terminus located extracellularly while the C-terminus is cytoplasmic.Signal through G proteins

TABLE VI Motifs and Post-translational Modifications of 193P1E1BN-glycosylation site Number of matches: 3 1 246-249 NKSE (SEQ ID NO:63)2 316-319 NSSS (SEQ ID NO:64) 3 340-343 NLTD (SEQ ID NO:65) cAMP- andcGMP-dependent protein kinase phosphorylation site 107-110 KKNS (SEQ IDNO:66) Protein kinase C phosphorylation site Number of matches: 10 122-24 TAR 2 53-55 TLK 3 103-105 SPR 4 152-154 SPR 5 149-151 SEK 6103-105 SPR 7 152-154 SPR 8 203-205 TPK 9 217-219 TPK 10 203-205 TPKCasein kinase II phosphorylation site Number of matches: 12 1 16-19 STLD(SEQ ID NO:67) 2 34-37 SDFE (SEQ ID NO:68) 3 53-56 TLKD (SEQ ID NO:69) 4110-113 SVHE (SEQ ID NO:70) 5 119-122 SDPE (SEQ ID NO:71) 6 124-127 SNCE(SEQ ID NO:72) 7 276-279 SDAE (SEQ ID NO:73) 8 318-321 SSND (SEQ IDNO:74) 9 336-339 TCFE (SEQ ID NO:75) 10 350-353 SSYE (SEQ ID NO:76) 11360-363 TPPE (SEQ ID NO:77) 12 408-411 SNKE (SEQ ID NO:78)N-myristoylation site 239-244 GLKNAR (SEQ ID NO:79)

TABLE VII Search Peptides variant 1: 9-mers, 10-mers and 15-mers (SEQ IDNO: 80) MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNIPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNLLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH GQNIRDVSNKEN variant5: 9-mers PVASSCISEKSPRSPQL (SEQ ID NO: 81) 10-mers PPVASSCISEKSPRSPQLS(SEQ ID NO: 82) 15-mers DDLSDPPVASSCISEKSPRSPQLSDFGLE (SEQ ID NO: 83)Variant 6: 9-mers NKSEEAIDAESRLND NV (SEQ ID NO: 84) 10-mersNNKSEEAIDAESRLND NVF (SEQ ID NO: 85) 15-mers LKNARNNKSEEAIDAESRLNDNVFATPSP (SEQ ID NO: 86) Variant 10: 9-mers KIPEDILQKFQWIYPTQKLNKMR (SEQID NO: 87) 10-mers TKIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 88) 15-mersTPPEVTKIPEDILQKFQWIYPTQKLNKMR (SEQ ID NO: 89) Variant 12: 9-mersRALDGEESLLSKYNSN (SEQ ID NO: 90) 10-mers QRALDGEESLLSKYNSNL (SEQ ID NO:91) 15-mers ETARLQRALDGEESLLSKYNSNLATPIA (SEQ ID NO: 92)Tables VIII-XXI:

TABLE VIII Start Subsequence Score V1-HLA-A1-9mers-193P1EIB Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 19 DCETARLQR 45.000 98LSDFGLERY 37.500 341 LTDPSSPTI 25.000 132 GIDFIKATK 20.000 78 LSDPPVASS15.000 129 NQEGIDFIK 13.500 272 QLEKSDAEY 9.000 28 ALDGEESDF 5.000 253DTESRLNDN 2.250 31 GEESDFEDY 2.250 33 ESDFEDYPM 1.500 306 VSTNYPLSK1.500 63 LSNCENFQK 1.500 225 ISEYTMCLN 1.350 37 EDYPMRILY 1.250 228YTMCLNEDY 1.250 319 SNDLEVEDR 1.250 71 KTDVKDDLS 1.250 232 LNEDYTMGL1.125 358 TPTPPEVTK 1.000 215 CVTPKLEHF 1.000 389 KAVPPSKRF 1.000 17TLDCETARL 1.000 277 DAEYTNSPL 0.900 321 DLEVEDRTS 0.900 323 EVEDRTSLV0.900 344 PSSPTISSY 0.750 349 ISSYENLLR 0.750 333 NSDTCFENL 0.750 275KSDAEYTNS 0.750 48 HSEVQTLKD 0.675 233 NEDYTMGLK 0.500 382 LATPIAIKA0.500 281 TNSPLVPTF 0.500 251 AIDTESRLN 0.500 370 DILQLLSKY 0.500 263FATPSPIIQ 0.500 302 SIALVSTNY 0.500 97 QLSDFGLER 0.500 219 KLEHFGISE0.450 381 NLATPIAIK 0.400 236 YTMGLKNAR 0.250 16 STLDCETAR 0.250 391VPPSKRFLK 0.250 267 SPIIQQLEK 0.250 209 KMDDFECVT 0.250 121 LLDKARLEN0.250 189 VTPPTKQSL 0.250 60 IPELSNCEN 0.225 367 IPEDILQLL 0.225 171VHEQEAINS 0.225 35 DFEDYPMRI 0.225 142 LMEKNSMDI 0.225 182 YKEEPVIVT0.225 175 EAINSDNYK 0.200 201 LKTPKCALK 0.200 110 QVLPNPPQA 0.200 83VASSCISGK 0.200 102 GLERYIVSQ 0.180 146 NSMDIMKIR 0.150 173 EQEAINSDN0.135 247 KSEEAIDTE 0.135 290 CTPGLKIPS 0.125 147 SMDIMKIRE 0.125 264ATPSPIIQQ 0.125 30 DGEESDFED 0.113 86 SCISGKSPR 0.100 330 LVLNSDTCF0.100 188 IVTPPTKQS 0.100 118 AVNLLDKAR 0.100 205 KCALKMDDF 0.100 160YGYSPRVKK 0.100 137 KATKVLMEK 0.100 390 AVPPSKRFL 0.100 126 RLENQEGID0.090 183 KEEPVIVTP 0.090 65 NCENFQKTD 0.090 212 DFECVTPKL 0.090 12RSLASTLDC 0.075 178 NSDNYKEEP 0.075 5 RSFCGKLRS 0.075 316 NSSSNDLEV0.075 350 SSYENLLRT 0.075 195 QSLVKVLKT 0.075 194 KQSLVKVLK 0.060 287PTFCTPGLK 0.050 57 DVNIPELSN 0.050 112 LPNPPQAVN 0.050 280 YTNSPLVPT0.050 106 YIVSQVLPN 0.050 224 GISEYTMCL 0.050 154 REYFQKYGY 0.050 257RLNDNVFAT 0.050 369 EDILQLLSK 0.050 55 KDDVNIPEL 0.050 152 KIREYFQKY0.050 366 KIPEDILQL 0.050 67 ENFQKTDVK 0.050 75 KDDLSDPPV 0.050 214ECVTPKLEH 0.050 V5-HLA-A1-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 2 VASSCISEK 0.200 5 SCISEKSPR 0.100 6 CISEKSPRS0.020 3 ASSCISEKS 0.015 7 ISEKSPRSP 0.014 9 EKSPRSPQL 0.010 4 SSCISEKSP0.002 1 PVASSCISE 0.001 8 SEKSPRSPQ 0.000 V6-HLA-A1-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 8 DAESRLNDN 0.900 6 AIDAESRLN0.500 2 KSEEAIDAE 0.135 3 SEEAIDAES 0.090 5 EAIDAESRL 0.010 4 EEAIDAESR0.050 1 NKSEEAIDA 0.003 7 IDAESRLND 0.000 9 AESRLNDNV 0.000V10-HLA-A1-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 2 IPEDILQKF 2.250 1 KIPEDILQK 1.000 12 WIYPTQKLN 0.100 13IYPTQKLNK 0.050 6 ILQKFQWIY 0.050 5 DILQKFQWI 0.010 10 FQWIYPTQK 0.003 3PEDILQKFQ 0.003 15 PTQKLNKMR 0.003 4 EDILQKFQW 0.003 14 YPTQKLNKM 0.0038 QKFQWIYPT 0.001 9 KFQWIYPTQ 0.001 11 QWIYPTQKL 0.001 7 LQKFQWIYP 0.000V12-HLA-A1-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 4 DGEESLLSK 22.500 5 GEESLLSKY 2.250 2 ALDGEESLL 0.500 7ESLLSKYNS 0.030 8 SLLSKYNSN 0.010 1 RALDGEESL 0.010 3 LDGEESLLS 0.003 6EESLLSKYN 0.001

TABLE IX Start Subsequence Score V1-HLA-A1-10mers-193P1EIB Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 219 KLEHFGISEY 45.000 126RLENQEGIDF 45.000 33 ESDFEDYPMR 15.000 341 LTDPSSPTIS 12.500 147SMDIMKIREY 12.500 30 DGEESDFEDY 11.250 390 AVPPSKRFLK 10.000 78LSDPPVASSC 7.500 173 EQEAINSDNY 6.750 36 FEDYPMRILY 6.250 323 EVEDRTSLVL4.500 153 IREYFQKYGY 4.500 232 LNEDYTMGLK 4.500 60 IPELSNCENF 2.250 253DTESRLNDNV 2.250 277 DAEYTNSPLV 1.800 247 KSEEAIDTES 1.350 367IPEDILQLLS 1.125 357 RTPTPPEVTK 1.000 305 LVSTNYPLSK 1.000 62 ELSNCENFQK1.000 19 DCETARLQRA 0.900 321 DLEVEDRTSL 0.900 65 NCENFQKTDV 0.900 301NSIALVSTNY 0.750 333 NSDTCFENLT 0.750 178 NSDNYKEEPV 0.750 98 LSDFGLERYI0.750 225 ISEYTMCLNE 0.675 129 NQEGIDFIKA 0.675 71 KTDVKDDLSD 0.625 17TLDCETARLQ 0.500 263 FATPSPIIQQ 0.500 210 MDDFECVTPK 0.500 132GIDFIKATKV 0.500 348 TISSYENLLR 0.500 289 FCTPGLKIPS 0.500 280YTNSPLVPTF 0.500 97 QLSDFGLERY 0.500 248 SEEAIDTESR 0.450 368 PEDILQLLSK0.250 190 TPPTKQSLVK 0.250 189 VTPPTKQSLV 0.250 128 ENQEGIDFIK 0.250 251AIDTESRLND 0.250 46 DLHSEVQTLK 0.200 266 PSPIIQQLEK 0.150 85 SSCISGKSPR0.150 15 ASTLDCETAR 0.150 318 SSNDLEVEDR 0.150 271 QQLEKSDAEY 0.150 48HSEVQTLKDD 0.135 343 DPSSPTISSY 0.125 258 LNDNVFATPS 0.125 233NEDYTMGLKN 0.125 319 SNDLEVEDRT 0.125 380 SNLATPIAIK 0.100 188IVTPPTKQSL 0.100 185 EPVIVTPPTK 0.100 110 QVLPNPPQAV 0.100 214ECVTPKLEHF 0.100 27 RALDGEESDF 0.100 131 EGIDFIKATK 0.100 382 LATPIAIKAV0.100 329 SLVLNSDTCF 0.100 215 CVTPKLEHFG 0.100 117 QAVNLLDKAR 0.100 389KAVPPSKRFL 0.100 102 GLERYIVSQV 0.090 272 QLEKSDAEYT 0.090 337CFENLTDPSS 0.090 379 NSNLATPIAI 0.075 275 KSDAEYTNSP 0.075 94 RSPQLSDFGL0.075 349 ISSYENLLRT 0.075 282 NSPLVPTFCT 0.075 291 TPGLKIPSTK 0.050 331VLNSDTCFEN 0.050 54 LKDDVNIPEL 0.050 381 NLATPIAIKA 0.050 16 STLDCETARL0.050 44 LYDLHSEVQT 0.050 236 YTMGLKNARN 0.050 28 ALDGEESDFE 0.050 112LPNPPQAVNL 0.050 75 KDDLSDPPVA 0.050 286 VPTFCTPGLK 0.050 121 LLDKARLENQ0.050 170 SVHEQEAINS 0.050 231 CLNEDYTMGL 0.050 141 VLMEKNSMDI 0.050 74VKDDLSDPPV 0.050 150 IMKIREYFQK 0.050 120 NLLDKARLEN 0.050 209KMDDFECVTP 0.050 290 CTPGLKIPST 0.050 136 IKATKVLMEK 0.050 183KEEPVIVTPP 0.045 351 SYENLLRTPT 0.045 35 DFEDYPMRIL 0.045V5-HLA-A1-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:11;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 5 SSCISEKSPR 0.150 8 ISEKSPRSPQ 0.135 6 SCISEKSPRS 0.020 2PVASSCISEK 0.020 3 VASSCISEKS 0.010 10 EKSPRSPQLS 0.005 4 ASSCISEKSP0.002 7 CISEKSPRSP 0.001 1 PPVASSCISE 0.000 9 SEKSPRSPQL 0.000V6-HLA-A1-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 3 KSEEAIDAES 2.700 9 DAESRLNDNV 0.900 4 SEEAIDAESR 0.450 7AIDAESRLND 0.250 6 EAIDAESRLN 0.010 1 NNKSEEAIDA 0.001 10 AESRLNDNVF0.001 8 IDAESRLNDN 0.001 5 EEAIDAESRL 0.001 2 NKSEEAIDAE 0.000V10-HLA-A1-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 13 WIYPTQKLNK 10.000 1 TKIPEDILQK 0.500 6 DILQKFQWIY 0.500 3IPEDILQKFQ 0.225 2 KIPEDILQKF 0.100 15 YPTQKLNKMR 0.025 4 PEDILQKFQW0.013 10 KFQWIYPTQK 0.010 9 QKFQWIYPTQ 0.001 7 ILQKFQWIYP 0.001 14IYPTQKLNKM 0.001 12 QWIYPTQKLN 0.001 5 EDILQKFQWI 0.001 8 LQKFQWIYPT0.000 11 FQWIYPTQKL 0.000 V12-HLA-A1-10mers-193P1E1B Each peptide is aportion of SEQ ID NO:25; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. 5 DGEESLLSKY 11.250 3 ALDGEESLLS 2.500 4LDGEESLLSK 0.050 6 GEESLLSKYN 0.045 8 ESLLSKYNSN 0.015 9 SLLSKYNSNL0.010 2 RALDGEESLL 0.010 7 EESLLSKYNS 0.001 1 QRALDGEESL 0.001

TABLE X Start Subsequence Score V1-HLA-A0201-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 43 ILYDLHSEV 1551.288 257RLNDNVFAT 407.580 111 VLPNPPQAV 118.238 366 KIPEDILQL 96.947 209KMDDFECVT 48.131 374 LLSKYNSNL 36.316 340 NLTDPSSPT 30.553 304 ALVSTNYPL21.362 229 TMCLNEDYT 14.504 224 GISEYTMCL 12.043 17 TLDCETARL 8.545 10KLRSLASTL 5.682 140 KVLMEKNSM 5.629 199 KVLKTPKCA 5.629 39 YPMRILYDL5.459 295 KIPSTKNSI 5.021 278 AEYTNSPLV 4.328 46 DLHSEVQTL 3.685 261NVFATPSPI 3.378 348 TISSYENLL 2.937 322 LEVEDRTSL 2.895 329 SLVLNSDTC2.434 208 LKMDDFECV 2.319 383 ATPIAIKAV 2.222 390 AVPPSKRFL 2.056 110QVLPNPPQA 1.608 350 SSYENLLRT 1.468 66 CENFQKTDV 1.352 200 VLKTPKCAL1.271 280 YTNSPLVPT 1.095 283 SPLVPTFCT 1.044 314 KTNSSSNDL 1.038 207ALKMDDFEC 1.009 119 VNLLDKARL 0.877 270 IQQLEKSDA 0.856 142 LMEKNSMDI0.820 95 SPQLSDFGL 0.809 371 ILQLLSKYN 0.697 352 YENLLRTPT 0.667 254TESRLNDNV 0.663 133 IDFIKATKV 0.608 145 KNSMDIMKI 0.548 52 QTLKDDVNI0.536 189 VTPPTKQSL 0.504 231 CLNEDYTMG 0.458 316 NSSSNDLEV 0.454 190TPPTKQSLV 0.454 195 QSLVKVLKT 0.414 373 QLLSKYNSN 0.414 103 LERYIVSQV0.402 300 KNSIALVST 0.392 141 VLMEKNSMD 0.384 64 SNCENFQKT 0.379 114NPPQAVNLL 0.321 265 TPSPIIQQL 0.321 378 YNSNLATPI 0.313 158 QKYGYSPRV0.309 380 SNLATPIAI 0.252 202 KTPKCALKM 0.242 307 STNYPLSKT 0.238 286VPTFCTPGL 0.237 97 QLSDFGLER 0.232 223 FGISEYTMC 0.224 50 EVQTLKDDV0.224 20 CETARLQRA 0.222 230 MCLNEDYTM 0.204 130 QEGIDFIKA 0.184 328TSLVLNSDT 0.180 282 NSPLVPTFC 0.178 45 YDLHSEVQT 0.176 14 LASTLDCET0.176 7 FCGKLRSLA 0.149 36 FEDYPMRIL 0.144 367 IPEDILQLL 0.143 331VLNSDTCFE 0.139 75 KDDLSDPPV 0.135 131 EGIDEIKAT 0.131 128 ENQEGIDFI0.130 58 VNIPELSNC 0.127 12 RSLASTLDC 0.120 323 EVEDRTSLV 0.120 382LATPIAIKA 0.117 324 VEDRTSLVL 0.116 168 KNSVHEQEA 0.114 291 TPGLKIPST0.112 135 FIKATKVLM 0.110 106 YIVSQVLPN 0.108 341 LTDPSSPTI 0.099 55KDDVNIPEL 0.096 250 EAIDTESRL 0.091 9 GKLRSLAST 0.088 399 KHGQNIRDV0.078 117 QAVNLLDKA 0.078 298 STKNSIALV 0.078 320 NDLEVEDRT 0.077 232LNEDYTMGL 0.062 70 QKTDVKDDL 0.060 99 SDFGLERYI 0.059 354 NLLRTPTPP0.055 237 TMGLKNARN 0.054 X-V5-HLA-A0201-9mers-193P1E1B Each peptide isa portion of SEQ ID NO:11; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight. 6 CISEKSPRS 0.042 9 EKSPRSPQL 0.002 2VASSCISEK 0.001 3 ASSCISEKS 0.000 5 SCISEKSPR 0.000 4 SSCISEKSP 0.000 1PVASSCISE 0.000 8 SEKSPRSPQ 0.000 7 ISEKSPRSP 0.000V6-HLA-A0201-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 9 AESRLNDNV 0.663 5 EAIDAESRL 0.091 1 NKSEEAIDA 0.028 6 AIDAESRLN0.001 7 IDAESRLND 0.000 2 KSEEAIDAE 0.000 3 SEEAIDAES 0.000 8 DAESRLNDN0.000 4 EEAIDAESR 0.000 V10-HLA-A0201-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:21; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 5 DILQKFQWI 4.160 6 ILQKFQWIY 1.480 14YPTQKLNKM 0.343 12 WIYPTQKLN 0.151 8 QKFQWIYPT 0.088 1 KIPEDILQK 0.06810 FQWIYPTQK 0.058 11 QWIYPTQKL 0.003 7 LQKFQWIYP 0.001 2 IPEDILQKF0.000 9 KFQWIYPTQ 0.000 4 EDILQKFQW 0.000 3 PEDILQKFQ 0.000 15 PTQKLNKMR0.000 13 IYPTQKLNK 0.000 V12-HLA-A0201-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:25; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 2 ALDGEESLL 8.545 1 RALDGEESL 2.205 8SLLSKYNSN 0.414 3 LDGEESLLS 0.001 6 EESLLSKYN 0.001 5 GEESLLSKY 0.000 7ESLLSKYNS 0.000 4 DGEESLLSK 0.000

TABLE XI Start Subsequence Score V1-HLA-A0201-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 141 VLMEKNSMDI 269.051 366KIPEDILQLL 96.947 231 CLNEDYTMGL 87.586 373 QLLSKYNSNL 79.041 340NLTDPSSPTI 42.774 42 RILYDLHSEV 35.385 199 KVLKTPKCAL 24.206 110QVLPNPPQAV 22.517 102 GLERYIVSQV 10.238 322 LEVEDRTSLV 9.426 355LLRTPTPPEV 8.986 374 LLSKYNSNLA 8.446 13 SLASTLDCET 7.452 194 KQSLVKVLKT6.082 381 NLATPIAIKA 4.968 228 YTMCLNEDYT 4.747 16 STLDCETARL 4.501 132GIDFIKATKV 3.825 382 LATPIAIKAV 3.777 229 TMCLNEDYTM 3.588 188IVTPPTKQSL 3.178 285 LVPTFCTPGL 3.178 207 ALKMDDFECV 2.266 127LENQEGIDFI 2.138 162 YSPRVKKNSV 2.088 118 AVNLLDKARL 1.869 303IALVSTNYPL 1.866 51 VQTLKDDVNI 1.798 189 VTPPIKQSLV 1.642 206 CALKMDDFEC1.481 216 VTPKLEHFGI 1.429 261 NVFATPSPII 1.385 272 QLEKSDAEYT 1.285 130QEGIDFIKAT 1.266 45 YDLHSEVQTL 1.161 269 IIQQLEKSDA 1.161 389 KAVPPSKRFL1.142 157 FQKYGYSPRV 1.135 120 NLLDKARLEN 1.130 295 KIPSTKNSIA 0.980 332LNSDTCFENL 0.905 94 RSPQLSDFGL 0.809 331 VLNSDTCFEN 0.735 264 ATPSPIIQQL0.682 220 LEHFGISEYT 0.664 49 SEVQTLKDDV 0.663 223 FGISEYTMCL 0.641 109SQVLPNPPQA 0.504 315 TNSSSNDLEV 0.454 208 LKMDDFECVT 0.416 97 QLSDFGLERY0.344 282 NSPLVPTFCT 0.282 74 VKDDLSDPPV 0.269 290 CTPGLKIPST 0.238 5RSFCGKLRSL 0.237 112 LPNPPQAVNL 0.237 296 IPSTKNSIAL 0.237 152KIREYFQKYG 0.234 34 SDFEDYPMRI 0.220 54 LKDDVNIPEL 0.190 306 VSTNYPLSKT0.190 349 ISSYENLLRT 0.190 142 LMEKNSMDIM 0.180 281 TNSPLVPTFC 0.178 69FQKTDVKDDL 0.171 63 LSNCENFQKT 0.157 358 TPTPPEVTKI 0.157 99 SDFGLERYIV0.147 371 ILQLLSKYNS 0.127 276 SDAEYTNSPL 0.122 168 KNSVHEQEAI 0.117 257RLNDNVFATP 0.116 271 QQLEKSDAEY 0.115 304 ALVSTNYPLS 0.112 9 GKLRSLASTL0.110 327 RTSLVLNSDT 0.104 321 DLEVEDRTSL 0.103 346 SPTISSYENL 0.102 224GISEYTMCLN 0.097 178 NSDNYKEEPV 0.089 20 CETARLQRAL 0.083 133 IDFIKATKVL0.077 398 LKHGQNIRDV 0.076 57 DVNIPELSNC 0.075 329 SLVLNSDTCF 0.075 333NSDTCFENLT 0.074 372 LQLLSKYNSN 0.071 365 TKIPEDILQL 0.068 379NSNLATPIAI 0.068 43 ILYDLHSEVQ 0.067 209 KMDDFECVTP 0.062 129 NQEGIDFIKA0.061 378 YNSNLATPIA 0.061 328 TSLVLNSDTC 0.059 14 LASTLDCETA 0.057 354NLLRTPTPPE 0.055 77 DLSDPPVASS 0.053 111 VLPNPPQAVN 0.052 256 SRLNDNVFAT0.051 98 LSDFGLERYI 0.051 V5-HLA-A0201-10mers-193P1E1B Each peptide is aportion of SEQ ID NO:11; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. 9 SEKSPRSPQL 0.015 7 CISEKSPRSP 0.002 3VASSCISEKS 0.001 6 SCISEKSPRS 0.000 5 SSCISEKSPR 0.000 4 ASSCISEKSP0.000 2 PVASSCISEK 0.000 8 ISEKSPRSPQ 0.000 10 EKSPRSPQLS 0.000 1PPVASSCISE 0.000 V6-HLA-A0201-10mers-193P1E1B Each peptide is a portionof SEQ ID NO:13; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. 5 EEAIDAESRL 0.031 9 DAESRLNDNV 0.002 8 IDAESRLNDN0.002 1 NNKSEEAIDA 0.001 7 AIDAESRLND 0.001 10 AESRLNDNVF 0.001 3KSEEAIDAES 0.000 2 NKSEEAIDAE 0.000 6 EAIDAESRLN 0.000 4 SEEAIDAESR0.000 V10-HLA-A0201-10mers-193P1E1B Each peptide is a portion of SEQ IDNO:21; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. 11 FQWIYPTQKL 82.694 2 KIPEDILQKF 0.338 7 ILQKFQWIYP 0.237 8LQKFQWIYPT 0.103 6 DILQKFQWIY 0.033 13 WIYPTQKLNK 0.030 5 EDILQKFQWI0.011 14 IYPTQKLNKM 0.003 15 YPTQKLNKMR 0.000 3 IPEDILQKFQ 0.000 9QKFQWIYPTQ 0.000 4 PEDILQKFQW 0.000 1 TKIPEDILQK 0.000 10 KFQWIYPTQK0.000 12 QWIYPTQKLN 0.000 V12-HLA-A0201-10mers-193P1E1B Each peptide isa portion of SEQ ID NO:25; each start position is specified, the lengthof peptide is 10 amino acids, and the end position for each peptide isthe start position plus nine. 9 SLLSKYNSNL 79.041 2 RALDGEESLL 4.501 3ALDGEESLLS 0.030 1 QRALDGEESL 0.001 6 GEESLLSKYN 0.001 4 LDGEESLLSK0.000 8 ESLLSKYNSN 0.000 7 EESLLSKYNS 0.000 5 DGEESLLSKY 0.000

TABLE XII Start Subsequence Score V1-HLA-A3-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 239 GLKNARNNK 60.000 381NLATPIAIK 45.000 97 QLSDFGLER 24.000 152 KIREYFQKY 16.200 132 GIDFIKATK9.000 129 NQEGIDFIK 4.050 397 FLKHGQNIR 4.000 272 QLEKSDAEY 4.000 387AIKAVPPSK 3.000 28 ALDGEESDF 3.000 194 KQSLVKVLK 2.700 137 KATKVLMEK2.700 304 ALVSTNYPL 2.700 197 LVKVLKTPK 2.000 374 LLSKYNSNL 1.800 10KLRSLASTL 1.800 224 GISEYTMCL 1.620 142 LMEKNSMDI 1.200 43 ILYDLHSEV1.000 209 KMDDFECVT 0.900 200 VLKTPKCAL 0.900 257 RLNDNVFAT 0.900 366KIPEDILQL 0.810 267 SPIIQQLEK 0.600 207 ALKMDDFEC 0.600 306 VSTNYPLSK0.600 391 VPPSKRFLK 0.600 302 SIALVSTNY 0.600 17 TLDCETARL 0.600 46DLHSEVQTL 0.540 358 TPTPPEVTK 0.450 236 YTMGLKNAR 0.450 215 CVTPKLEHF0.450 219 KLEHFGISE 0.360 186 PVIVTPPTK 0.300 83 VASSCISGK 0.300 111VLPNPPQAV 0.300 63 LSNCENFQK 0.300 330 LVLNSDTCF 0.300 16 STLDCETAR0.300 261 NVFATPSPI 0.300 228 YTMCLNEDY 0.300 329 SLVLNSDTC 3.000 2DPIRSFCGK 0.270 102 GLERYIVSQ 0.270 370 DILQLLSKY 0.270 118 AVNLLDKAR0.200 116 PQAVNLLDK 0.180 402 QNIRDVSNK 0.180 295 KIPSTKNSI 0.180 348TISSYENLL 0.180 154 REYFQKYGY 0.180 160 YGYSPRVKK 0.150 287 PTFCTPGLK0.150 340 NLTDPSSPT 0.150 149 DIMKIREYF 0.135 211 DDFECVTPK 0.135 157FQKYGYSPR 0.120 31 GEESDFEDY 0.108 389 KAVPPSKRF 0.101 229 TMCLNEDYT0.100 151 MKIREYFQK 0.090 140 KVLMEKNSM 0.090 314 KTNSSSNDL 0.090 205KCALKMDDF 0.090 293 GLKIPSTKN 0.090 175 EAINSDNYK 0.090 39 YPMRILYDL0.061 67 ENFQKTDVK 0.060 202 KTPKCALKM 0.060 150 IMKIREYFQ 0.060 86SCISGKSPR 0.060 53 TLKDDVNIP 0.060 47 LHSEVQTLK 0.045 199 KVLKTPKCA0.045 110 QVLPNPPQA 0.045 364 VTKIPEDIL 0.045 141 VLME KNSMD 0.045 52QTLKDDVNI 0.045 189 VTPPTKQSL 0.045 341 LTDPSSPTI 0.045 349 ISSYENLLR0.040 121 LLDKARLEN 0.040 147 SMDIMKIRE 0.040 191 PPTKQSLVK 0.040 196SLVKVLKTP 0.034 146 NSMDIMKIR 0.034 201 LKTPKCALK 0.030 231 CLNEDYTMG0.030 373 QLLSKYNSN 0.030 34 SDFEDYPMR 0.030 24 RLQRALDGE 0.030 98LSDFGLERY 0.030 354 NLLRTPTPP 0.030 355 LLRTPTPPE 0.030 13 SLASTLDCE0.030 363 EVTKIPEDI 0.027 369 EDILQLLSK 0.027 233 NEDYTMGLK 0.027 319SNDLEVEDR 0.024 V5-HLA-A3-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of pdptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 2 VASSCISEK 0.300 5 SCISEKSPR 0.060 6 CISEKSPRS0.006 1 PVASSCISE 0.000 3 ASSCISEKS 0.000 9 EKSPRSPQL 0.000 8 SEKSPRSPQ0.000 4 SSCISEKSP 0.000 7 ISEKSPRSP 0.000 V6-HLA-A3-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 4 EEAIDAESR 0.004 5 EAIDAESRL0.003 2 KSEEAIDAE 0.001 1 NKSEEAIDA 0.001 9 AESRLNDNV 0.001 6 AIDAESRLN0.000 3 SEEAIDAES 0.000 8 DAESRLNDN 0.000 7 IDAESRLND 0.000V10-HLA-A3-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 6 ILQKFQWIY 36.000 1 KIPEDILQK 27.000 10 FQWIYPTQK 9.000 5DILQKFQWI 0.081 2 IPEDILQKF 0.045 13 IYPTQKLNK 0.040 15 PTQKLNKMR 0.01012 WIYPTQKLN 0.007 8 QKFQWIYPT 0.007 14 YPTQKLNKM 0.003 11 QWIYPTQKL0.001 7 LQKFQWIYP 0.001 4 EDILQKFQW 0.000 9 KFQWIYPTQ 0.000 3 PEDILQKFQ0.000 V12-HLA-A3-9mers-193P1E1B Each peptide is a portion of SEQ IDNO:25; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 2 ALDGEESLL 0.900 5 GEESLLSKY 0.054 8 SLLSKYNSN 0.030 4DGEESLLSK 0.027 1 RALDGEESL 0.009 7 ESLLSKYNS 0.000 3 LDGEESLLS 0.000 6EESLLSKYN 0.000

TABLE XIII Start Subsequence Score V1-HLA-A3-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 150 IMKIREYFQK 60.000 196SLVKVLKTPK 30.000 200 VLKTPKCALK 20.000 219 KLEHFGISEY 18.000 62ELSNCENFQK 18.000 305 LVSTNYPLSK 12.000 46 DLHSEVQTLK 9.000 390AVPPSKRFLK 9.000 97 QLSDFGLERY 6.000 231 CLNEDYTMGL 5.400 401 GQNIRDVSNK5.400 126 RLENQEGIDF 4.000 329 SLVLNSDTCF 3.000 102 GLERYIVSQV 2.700 373QLLSKYNSNL 2.700 141 VLMEKNSMDI 2.700 357 RTPTPPEVTK 1.500 348TISSYENLLR 0.800 366 KIPEDILQLL 0.608 207 ALKMDDFECV 0.600 147 SMDIMKIREY 0.600 381 NLATPIAIKA 0.600 387 AK AVPPSKR 0.600 340 NLTDPSSPTI 0.600229 TMCLNEDYTM 0.600 386 IAIKAVPPSK 0.450 261 NVFATPSPII 0.450 199KVLKTPKCAL 0.405 190 TPPTKQSLVK 0.400 82 PVASSCISGK 0.300 291 TPGLKIPSTK0.300 142 LMEKNSMDIM 0.300 280 YTNSPLVPTF 0.300 271 QQLEKSDAEY 0.270 355LLRTPTPPEV 0.200 374 LLSKYNSNLA 0.200 321 DLEVEDRTSL 0.180 380SNLATPIAIK 0.135 143 MEKNSMDIMK 0.120 371 ILQLLSKYNS 0.120 239GLKNARNNKS 0.120 96 PQLSDFGLER 0.108 13 SLASTLDCET 0.100 43 ILYDLHSEVQ0.100 272 QLEKSDAEYT 0.100 159 KYGYSPRVKK 0.090 136 IKATKVLMEK 0.090 216VTPKLEHFGI 0.090 188 IVTPPTKQSL 0.090 257 RLNDNVFATP 0.900 185EPVIVTPPTK 0.090 128 ENQEGIDFIK 0.081 264 ATPSPIIQQL 0.068 110QVLPNPPQAV 0.068 1 MDPIRSFCGK 0.060 210 MDDFECVTPK 0.060 115 PPQAVNLLDK0.060 304 ALVSTNYPLS 0.060 295 KIPSTKNSIA 0.060 293 GLKIPSTKNS 0.060 53TLKDDVNIPE 0.060 118 AVNLLDKARL 0.600 132 GIDFIKATKV 0.060 331VLNSDTCFEN 0.060 174 QEAINSDNYK 0.060 66 CENFQKTDVK 0.060 10 KLRSLASTLD0.060 286 VPTFCTPGLK 0.060 285 LVPTFCTPGL 0.060 318 SSNDLEVEDR 0.060 209KMDDFECVTP 0.060 120 NLLDKARLEN 0.060 77 DLSDPPVASS 0.054 194 KQSLVKVLKT0.054 129 NQEGIDFIKA 0.054 151 MKIREYFQKY 0.054 301 NSIALVSTNY 0.045 354NLLRTPTPPE 0.045 16 STLDCETARL 0.045 287 PTFCTPGLKI 0.045 397 FLKHGQNIRD0.040 323 EVEDRTSLVL 0.036 173 EQEAINSDNY 0.036 193 TKQSLVKVLK 0.030 238MGLKNARNNK 0.030 28 ALDGEESDFE 0.030 42 RILYDLHSEV 0.030 117 QAVNLLDKAR0.030 27 RALDGEESDF 0.030 111 VLPNPPQAVN 0.030 92 SPRSPQLSDF 0.030 121LLDKARLENQ 0.030 358 TPTPPEVTKI 0.027 303 IALVSTNYPL 0.027 34 SDFEDYPMRI0.027 363 EVTKIPEDIL 0.027 145 KNSMDIMKIR 0.027 69 FQKTDVKDDL 0.027 36FEDYPMRILY 0.024 40 PMRILYDLHS 0.024 V5-HLA-A3-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:11; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 2 PVASSCISEK 0.300 5 SSCISEKSPR0.020 9 SEKSPRSPQL 0.002 6 SCISEKSPRS 0.001 3 VASSCISEKS 0.001 7CISEKSPRSP 0.000 8 ISEKSPRSPQ 0.000 1 PPVASSCISE 0.000 4 ASSCISEKSP0.000 10 EKSPRSPQLS 0.000 V6-HLA-A3-10mers-193P1E1B Each peptide is aportion of SEQ ID NO:13; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. 4 SEEAIDAESR 0.012 10 AESRLNDNVF 0.006 7AIDAESRLND 0.004 3 KSEEAIDAES 0.001 1 NNKSEEAIDA 0.001 9 DAESRLNDNV0.001 5 EEAIDAESRL 0.001 8 IDAESRLNDN 0.000 2 NKSEEAIDAE 0.000 6EAIDAESRLN 0.000 V10-HLA-A3-10mers-193P1E1B Each peptide is a portion ofSEQ ID NO:21; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. 13 WIYPTQKLNK 30.000 2 KIPEDILQKF 2.025 6 DILQKFQWIY1.620 10 KFQWIYPTQK 0.180 1 TKIPEDILQK 0.135 11 FQWIYPTQKL 0.135 8LQKFQWIYPT 0.041 7 ILQKFQWIYP 0.040 15 YPTQKLNKMR 0.020 5 EDILQKFQWI0.001 14 IYPTQKLNKM 0.000 4 PEDILQKFQW 0.000 9 QKFQWIYPTQ 0.000 3IPEDILQKFQ 0.000 12 QWIYPTQKLN 0.000 V12-HLA-A3-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:25; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 9 SLLSKYNSNL 2.700 3 ALDGEESLLS0.120 4 LDGEESLLSK 0.090 2 RALDGEESLL 0.009 5 DGEESLLSKY 0.003 1QRALDGEESL 0.001 7 EESLLSKYNS 0.000 6 GEESLLSKYN 0.000 8 ESLLSKYNSN0.000

TABLE XIV Start Subsequence Score V1-HLA-A1101-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 197 LVKVLKTPK 2.000 194KQSLVKVLK 1.800 129 NQEGIDFIK 1.800 239 GLKNARNNK 1.200 137 KATKVLMEK1.200 132 GIDFIKATK 1.200 391 VPPSKRFLK 0.600 267 SPIIQQLEK 0.600 387AIKAVPPSK 0.400 236 YTMGLKNAR 0.400 381 NLATPIAIK 0.400 186 PVIVTPPTK0.300 16 STLDCETAR 0.300 118 AVNLLDKAR 0.200 287 PTFCTPGLK 0.200 358TPTPPEVTK 0.200 83 VASSCISGK 0.200 97 QLSDFGLER 0.160 116 PQAVNLLDK0.120 159 KYGYSPRVK 0.120 157 FQKYGYSPR 0.120 151 MKIREYFQK 0.090 140KVLMEKNSM 0.090 175 EAINSDNYK 0.090 2 DPIRSFCGK 0.090 397 FLKHGQNIR0.080 402 QNIRDVSNK 0.060 63 LSNCENFQK 0.060 202 KTPKCALKM 0.060 233NEDYTMGLK 0.060 86 SCISGKSPR 0.060 199 KVLKTPKCA 0.045 160 YGYSPRVKK0.040 191 PPTKQSLVK 0.040 306 VSTNYPLSK 0.040 261 NVFATPSPI 0.040 330LVLNSDTCF 0.030 110 QVLPNPPQA 0.030 314 KTNSSSNDL 0.030 67 ENFQKTDVK0.024 224 GISEYTMCL 0.024 19 DCETARLQR 0.024 366 KIPEDILQL 0.024 47LHSEVQTLK 0.020 228 YTMCLNEDY 0.020 215 CVTPKLEHF 0.020 201 LKTPKCALK0.020 369 EDILQLLSK 0.018 52 QTLKDDVNI 0.015 295 KIPSTKNSI 0.012 144EKNSMDIMK 0.012 211 DDFECVTPK 0.012 304 ALVSTNYPL 0.012 10 KLRSLASTL0.012 152 KIREYFQKY 0.012 298 STKNSIALV 0.010 189 VTPPTKQSL 0.010 341LTDPSSPTI 0.010 364 VTKIPEDIL 0.010 396 RFLKHGQNI 0.009 142 LMEKNSMDI0.008 349 ISSYENLLR 0.008 34 SDFEDYPMR 0.008 319 SNDLEVEDR 0.008 43ILYDLHSEV 0.008 39 YPMRILYDL 0.008 154 REYFQKYGY 0.007 323 EVEDRTSLV0.006 165 RVKKNSVHE 0.006 50 EVQTLKDDV 0.006 363 EVTKIPEDI 0.006 205KCALKMDDF 0.006 217 TPKLEHFGI 0.006 95 SPQLSDFGL 0.006 270 IQQLEKSDA0.006 230 MCLNEDYTM 0.006 383 ATPIAIKAV 0.005 389 KAVPPSKRF 0.005 200VLKTPKCAL 0.004 146 NSMDIMKIR 0.004 382 LATPIAIKA 0.004 222 HFGISEYTM0.004 272 QLEKSDAEY 0.004 28 ALDGEESDF 0.004 111 VLPNPPQAV 0.004 288TFCTPGLKI 0.004 135 FIKATKVLM 0.004 388 IKAVPPSKR 0.004 17 TLDCETARL0.004 374 LLSKYNSNL 0.004 348 TISSYENLL 0.004 181 NYKEEPVIV 0.004 302SIALVSTNY 0.004 257 RLNDNVFAT 0.004 249 EEAIDTESR 0.004 292 PGLKIPSTK0.003 71 KTDVKDDLS 0.003 357 RTPTPPEVT 0.003 117 QAVNLLDKA 0.003 327RTSLVLNSD 0.003 V5-HLA-A1101-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 2 VASSCISEK 0.200 5 SCISEKSPR 0.060 1 PVASSCISE0.000 6 CISEKSPRS 0.000 9 EKSPRSPQL 0.000 8 SEKSPRSPQ 0.000 3 ASSCISEKS0.000 4 SSCISEKSP 0.000 7 ISEKSPRSP 0.000 V6-HLA-A1101-9mers-193P1E1BEach peptide is a portion of SEQ ID NO:13; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plus eight. 4 EEAIDAESR 0.004 5EAIDAESRL 0.001 9 AESRLNDNV 0.001 1 NKSEEAIDA 0.000 2 KSEEAIDAE 0.000 8DAESRLNDN 0.000 3 SEEAIDAES 0.000 7 IDAESRLND 0.000 6 AIDAESRLN 0.000V10-HLA-A1101-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 1 KIPEDILQK 2.400 10 FQWIYPTQK 1.200 13 IYPTQKLNK 0.800 15PTQKLNKMR 0.010 6 ILQKFQWIY 0.008 14 YPTQKLNKM 0.002 2 IPEDILQKF 0.002 5DILQKFQWI 0.002 7 LQKFQWIYP 0.001 9 KFQWIYPTQ 0.001 12 WIYPTQKLN 0.00011 QWIYPTQKL 0.000 4 EDILQKFQW 0.000 8 QKFQWIYPT 0.000 3 PEDILQKFQ 0.000V12-HLA-A1101-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 4 DGEESLLSK 0.012 1 RALDGEESL 0.009 2 ALDGEESLL 0.004 5 GEESLLSKY0.002 8 SLLSKYNSN 0.001 3 LDGEESLLS 0.000 7 ESLLSKYNS 0.000 6 EESLLSKYN0.000

TABLE XV Start Subsequence Score V1-HLA-A11-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 390 AV PPSKRFLK 6.000 305LVSTNYPLSK 4.000 357 TPTP PEVTK 3.000 401 GQNIRDVSNK 1.800 159KYGYSPRVKK 1.200 150 IMKIREYFQK 1.200 196 SLVKVLKTPK 0.600 200VLKTPKCALK 0.400 190 TPPTKQ SLVK 0.400 62 ELSNCENFQK 0.360 386IAIKAVPPSK 0.300 291 TPGLKIPSTK 0.200 286 VPTFCTPGL K 0.200 82PVASSCISGK 0.200 396 RFLKHGQ NIR 0.180 348 TISSYENLLR 0.160 46DLHSEVQTLK 0.120 143 MEKNSMDIMK 0.120 199 KVLKTPKCAL 0.090 185EPVIVTPPTK 0.090 387 AIKAVPPSKR 0.080 380 SNLATPIAIK 0.060 174 QEAINSDNYK 0.060 66 CENFQKTDVK 0.060 136 IKATKVLMEK 0.040 115 PPQAVNLLDK0.040 156 YFQKYGYSPR 0.040 261 NVFATPSPII 0.040 232 LNEDYTMGLK 0.040 96PQLSDFGLER 0.036 128 ENQEGIDFIK 0.036 238 MGLKNARNNK 0.030 110QVLPNPPQAV 0.030 117 QAVNLLDKAR 0.030 216 VTPKLEHFGI 0.030 235DYTMGLKNAR 0.024 126 RLENQEGIDF 0.024 188 IVTPPTKQSL 0.020 193TKQSLVKVLK 0.020 1 MDPIRSFCGK 0.020 210 MDDFECVTPK 0.020 118 AVNLLDKARL0.020 285 LVPTFCTPGL 0.020 42 RILYDLHSEV 0.018 141 VLMEKNSMDI 0.016 16STLDCETARL 0.015 145 KNSMDIMKIR 0.012 129 NQEGIDFIKA 0.012 219KLEHFGISEY 0.012 132 GIDFIKATKV 0.012 368 PEDILQLLSK 0.012 366KIPEDILQLL 0.012 295 KIPSTKNSIA 0.012 323 EVEDRTSLVL 0.012 248SEEAIDTESR 0.012 102 GLERYIVSQV 0.012 377 KYNSNLATPI 0.012 264ATPSPIIQQL 0.010 280 YTNSPLVPTF 0.010 189 VTPPT KQSLV 0.010 27RALDGEESDF 0.009 131 EGIDFIKATK 0.009 140 KVLMEKNSMD 0.009 109SQVLPNPPQA 0.009 271 QQLEKSDAEY 0.009 231 CLNEDYTMGL 0.008 18 LDCETARLQR0.008 381 NLATPIAIKA 0.008 229 TMCLNEDYTM 0.008 51 VQTLKDDVNI 0.006 373QLLSK YNSNL 0.006 69 FQKTDVKDDL 0.006 303 IALVSTNYPL 0.006 329SLVLNSDTCF 0.006 124 KARLENQEGI 0.006 363 EVTKIPEDIL 0.006 71 KTDVKDDLSD0.006 157 FQKYGYSPRV 0.006 165 RVKKNSVHEQ 0.006 158 QKYGYSPRVK 0.004 266PSPIIQQLEK 0.004 269 IIQQLEKSDA 0.004 287 PTFCTPGLKI 0.004 85 SSCISGKSPR0.004 39 YPMRILYDLH 0.004 374 LLSKYNSNLA 0.004 355 LLRTPTPPEV 0.004 3PIRSFCGKLR 0.004 296 IPSTKNSIAL 0.004 15 ASTLDCETAR 0.004 97 QLSDFGLERY0.004 207 ALKMDDFECV 0.004 170 SVHEQEAINS 0.004 318 SSNDLEVEDR 0.004 142LMEKNSMDIM 0.004 391 VPPSKRFLKH 0.004 340 NLTDPSSPTI 0.004 194KQSLVKVLKT 0.004 105 RYIVSQVLPN 0.004 314 KTNSSSNDLE 0.003V5-HLA-A1101-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:11,each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 2 PVASSCISEK 0.200 5 SSCISEKSPR 0.004 9 SEKSPRSPQL 0.001 6SCISEKSPRS 0.000 3 VASSCISEKS 0.000 1 PPVASSCISE 0.000 7 CISEKSPRSP0.000 8 ISEKSPRSPQ 0.000 4 ASSCISEKSP 0.000 10 EKSPRSPQLS 0.000V6-HLA-A1101-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13,each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 4 SEEAIDAESR 0.012 1 NNKSEEAIDA 0.001 7 AIDAESRLND 0.001 9DAESRLNDNV 0.001 10 AESRLNDNVF 0.001 5 EEAIDAESRL 0.000 3 KSEEAIDAES0.000 8 IDAESRLNDN 0.000 2 NKSEEAIDAE 0.000 6 EAIDAESRLN 0.000V10-HLA-A1101-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 13 WIYPTQKLNK 1.600 10 KFQWIYPTQK 0.600 1 TKIPEDILQK 0.060 15YPTQKLNKMR 0.020 2 KIPEDILQKF 0.012 11 FQWIYPTQKL 0.012 14 IYPTQKLNKM0.004 6 DILQKFQWIY 0.004 8 LQKFQWIYPT 0.001 7 ILQKFQWIYP 0.001 4PEDILQKFQW 0.000 3 IPEDILQKFQ 0.000 5 EDILQKFQWI 0.000 9 QKFQWIYPTQ0.000 12 QWIYPTQKLN 0.000 V12-HLA-A1101-10mers-193P1E1B Each peptide isa portion of SEQ ID NO:25; each start position is specified, the lengthof peptide is 10 amino acids, and the end position for each peptide isthe start position plus nine. 4 LDGEESLLSK 0.040 2 RALDGEESLL 0.009 9SLLSKYNSNL 0.006 3 ALDGEESLLS 0.001 1 QRALDGEESL 0.000 6 GEESLLSKYN0.000 5 DGEESLLSKY 0.000 7 EESLLSKYNS 0.000 8 ESLLSKYNSN 0.000

TABLE XVI Start Subsequence Score V1-HLA-A24-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 212 DFECVTPKL 46.200 134DFIKATKVL 30.000 6 SFCGKLRSL 20.000 396 RFLKHGQNI 18.000 366 KIPEDILQL14.400 314 KTNSSSNDL 14.400 367 IPEDILQLL 12.096 10 KLRSLASTL 9.600 35DFEDYPMRI 9.000 189 VTPPTKQSL 8.640 39 YPMRILYDL 8.400 265 TPSPIIQQL8.064 309 NYPLSKTNS 7.500 114 NPPQAVNLL 7.200 250 EAIDTESRL 7.200 389KAVPPSKRF 7.200 390 AVPPSKRFL 7.200 232 LNEDYTMGL 7.200 161 GYSPRVKKN6.600 95 SPQLSDFGL 6.000 119 VNLLDKARL 6.000 304 ALVSTNYPL 6.000 277DAEYTNSPL 6.000 181 NYKEEPVIV 6.000 288 TFCTPGLKI 5.500 235 DYTMGLKNA5.000 262 VFATPSPII 5.000 155 EYFQKYGYS 5.000 224 GISEYTMCL 4.800 46DLHSEVQTL 4.800 21 ETARLQRAL 4.800 333 NSDTCFENL 4.800 348 TISSYENLL4.800 149 DIMKIREYF 4.200 200 VLKTPKCAL 4.000 286 VPTFCTPGL 4.000 364VTKIPEDIL 4.000 17 TLDCETARL 4.000 205 KCALKMDDF 4.000 374 LLSKYNSNL4.000 295 KIPSTKNSI 3.600 330 LVLNSDTCF 3.000 244 RNNKSEEAI 3.000 281TNSPLVPTF 2.880 222 HFGISEYTM 2.500 215 CVTPKLEHF 2.400 255 ESRLNDNVF2.400 145 KNSMDIMKI 2.200 28 ALDGEESDF 2.000 128 ENQEGIDFI 1.800 140KVLMEKNSM 1.800 202 KTPKCALKM 1.650 380 SNLATPIAI 1.500 105 RYIVSQVLP1.500 142 LMEKNSMDI 1.500 169 NSVHEQEAI 1.500 80 DPPVASSCI 1.500 52QTLKDDVNI 1.500 377 KYNSNLATP 1.500 363 EVTKIPEDI 1.400 341 LTDPSSPTI1.200 378 YNSNLATPI 1.200 180 DNYKEEPVI 1.000 217 TPKLEHFGI 1.000 159KYGYSPRVK 1.000 261 NVFATPSPI 1.000 337 CFENLTDPS 0.900 351 SYENLLRTP0.900 55 KDDVNIPEL 0.880 230 MCLNEDYTM 0.750 38 DYPMRILYD 0.750 322LEVEDRTSL 0.720 193 TKQSLVKVL 0.720 113 PNPPQAVNL 0.720 104 ERYIVSQVL0.672 70 QKTDVKDDL 0.672 227 EYTMCLNED 0.660 347 PTISSYENL 0.600 33ESDFEDYPM 0.500 44 LYDLHSEVQ 0.500 135 FIKATKVLM 0.500 279 EYTNSPLVP0.500 100 DFGLERYIV 0.500 90 GKSPRSPQL 0.480 3 PIRSFCGKL 0.440 324VEDRTSLVL 0.400 297 PSTKNSIAL 0.400 36 FEDYPMRIL 0.400 152 KIREYFQKY0.380 91 KSPRSPQLS 0.360 257 RLNDNVFAT 0.360 199 KVLKTPKCA 0.300 357RTPTPPEVT 0.300 12 RSLASTLDC 0.300 127 LENQEGIDF 0.300 168 KNSVHEQEA0.264 209 KMDDFECVT 0.240 185 EPVIVTPPT 0.210 282 NSPLVPTFC 0.210 400HGQNIRDVS 0.210 V5-HLA-A24-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 9 EKSPRSPQL 0.480 3 ASSCISEKS 0.154 6 CISEKSPRS0.120 7 ISEKSPRSP 0.015 5 SCISEKSPR 0.015 2 VASSCISEK 0.011 4 SSCISEKSP0.010 8 SEKSPRSPQ 0.001 1 PVASSCISE 0.001 V6-HLA-A24-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 5 EAIDAESRL 7.200 8 DAESRLNDN0.180 6 AIDAESRLN 0.100 2 KSEEAIDAE 0.036 3 SEEAIDAES 0.023 9 AESRLNDNV0.012 1 NKSEEAIDA 0.012 7 IDAESRLND 0.001 4 EEAIDAESR 0.001V10-HLA-A24-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 11 QWIYPTQKL 7.920 2 IPEDILQKF 6.653 5 DILQKFQWI 2.160 13IYPTQKLNK 0.750 14 YPTQKLNKM 0.660 9 KFQWIYPTQ 0.210 6 ILQKFQWIY 0.15012 WIYPTQKLN 0.120 1 KIPEDILQK 0.036 4 EDILQKFQW 0.015 7 LQKFQWIYP 0.0108 QKFQWIYPT 0.010 10 FQWIYPTQK 0.010 15 PTQKLNKMR 0.002 3 PEDILQKFQ0.000 V12-HLA-A24-9mers-193P1E1B Each peptide is a portion of SEQ IDNO:25; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 1 RALDGEESL 14.400 2 ALDGEESLL 4.000 8 SLLSKYNSN 0.180 7ESLLSKYNS 0.150 5 GEESLLSKY 0.020 4 DGEESLLSK 0.018 6 EESLLSKYN 0.012 3LDGEESLLS 0.012

TABLE XVII Start Subsequence Score V1-HLA-A24-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 38 DYPMRILYDL 420.000 377KYNSNLATPI 180.000 35 DFEDYPMRIL 36.000 366 KIPEDILQLL 24.192 105RYIVSQVLPN 15.000 389 KAVPPSKRFL 14.400 94 RSPQLSDFGL 12.000 199KVLKTPKCAL 12.000 264 ATPSPIIQQL 10.080 351 SYENLLRTPT 9.000 309NYPLSKTNSS 9.000 161 GYSPRVKKNS 8.400 5 RSFCGKLRSL 8.000 231 CLNEDYTMGL7.200 16 STLDCETARL 7.200 112 LPNPPQAVNL 7.200 323 EV EDRTSLVL 7.200 27RALDGEESDF 7.200 2 DPIRSFCGKL 6.600 227 EYTMCLNEDY 6.000 181 NYKEEPVIVT6.000 321 DLEVEDRTSL 6.000 118 AVNLL DKARL 6.000 373 QLLSKYNSNL 6.000126 RLENQ EGIDF 6.000 285 LVPTFCTPGL 6.000 223 FGISEYTMCL 6.000 303IALVSTNYPL 6.000 188 IVTPPTKQSL 5.760 332 LNSDTCFENL 5.760 69 FQKTDVKDDL5.600 44 LYDLHSEVQT 5.000 279 EYTNSPLVPT 5.000 296 IPSTKNSIAL 4.000 363EVTKIPEDIL 4.000 89 SGKSPRSPQL 4.000 346 SPTISSYENL 4.000 134 DFIKATKVLM3.750 280 YTNSPLVPTF 3.600 60 IPELSNCENF 3.000 214 ECVTPKLEHF 3.000 329SLVLN SDTCF 3.000 124 KARL ENQEGI 2.000 92 SPRSPQLSDF 2.000 168 KNSVHEQEAI 2.000 141 VLMEKNSMDI 1.800 260 DNVFATPSPI 1.500 379 NSNLATPIAI1.500 216 VTPKLEHFGI 1.500 358 TPTPPEVTKI 1.320 340 NLTDPSSPTI 1.200 98LSDFGLERYI 1.200 159 KYGYSPRVKK 1.100 51 VQTLKDDVNI 1.000 261 NVFATPSPII1.000 113 PNPPQAVNLL 0.864 337 CFENLTDPSS 0.750 142 LMEKNSMDIM 0.750 211DDFECVTPKL 0.739 9 GKLRSLASTL 0.720 365 TKIPEDILQL 0.720 347 PTISSYENLL0.720 45 YDLHSEVQTL 0.720 235 DYTMGLKNAR 0.720 103 LERYIVSQVL 0.672 6SFCGKLRSLA 0.600 247 KSEEAIDTES 0.554 54 LKDDVNIPEL 0.528 155 EYFQKYGYSP0.500 222 HFGISEYTMC 0.500 100 DFGLERYIVS 0.500 229 TMCLNEDYTM 0.500 276SDAEYTNSPL 0.480 20 CETARLQRAL 0.480 192 PTKQ SLVKVL 0.480 313SKTNSSSNDL 0.480 148 MDIMKIREYF 0.420 133 IDFIKATKVL 0.400 249EEAIDTESRL 0.400 42 RILYDLHSEV 0.396 219 KLEHFGISEY 0.330 295 KIPSTKNSIA0.300 137 KATKVLMEKN 0.264 300 KNSIALVSTN 0.240 254 TESRLNDNVF 0.240 395KRFLK HGQNI 0.240 327 RTSLVL NSDT 0.240 63 LSNCENFQKT 0.238 194KQSLVKVLKT 0.220 294 LKIPSTKNSI 0.216 110 QVLPNPPQAV 0.216 367IPEDILQLLS 0.216 30 DGEESDFEDY 0.216 102 GLERYIVSQV 0.210 301 NSIALVSTNY0.210 388 IKAVPPSKRF 0.200 271 QQLEKSDAEY 0.198 59 NIPELSNCEN 0.198 129NQEGIDFIKA 0.198 120 NLLDKARLEN 0.198 V5-HLA-A24-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:11; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 9 SEKSPRSPQL 0.400 3 VASSCISEKS0.154 6 SCISEKSPRS 0.150 8 ISEKSPRSPQ 0.015 10 EKSPRSPQLS 0.014 7CISEKSPRSP 0.012 4 ASSCISEKSP 0.010 5 SSCISEKSPR 0.010 1 PPVASSCISE0.002 2 PVASSCISEK 0.001 V6-HLA-A24-10mers-193P1E1B Each peptide is aportion of SEQ ID NO:13; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. 3 KSEEAIDAES 0.554 5 EEAIDAESRL 0.400 10AESRLNDNVF 0.240 6 EAIDAESRLN 0.180 9 DAESRLNDNV 0.180 1 NNKSEEAIDA0.100 8 IDAESRLNDN 0.014 7 AIDAESRLND 0.010 4 SEEAIDAESR 0.002 2NKSEEAIDAE 0.001 V10-HLA-A24-10mers-193P1E1B Each peptide is a portionof SEQ ID NO:21; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. 14 IYPTQKLNKM 49.500 2 KIPEDILQKF 13.306 11FQWIYPTQKL 5.280 5 EDILQKFQWI 0.216 10 KFQWIYPTQK 0.150 6 DILQKFQWIY0.150 12 QWIYPTQKLN 0.150 8 LQKFQWIYPT 0.100 3 IPEDILQKFQ 0.022 7ILQKFQWIYP 0.015 15 YPTQKLNKMR 0.012 13 WIYPTQKLNK 0.012 1 TKIPEDILQK0.002 9 QKFQWIYPTQ 0.001 4 PEDILQKFQW 0.001 V12-HLA-A24-10mers-193P1E1BEach peptide is a portion of SEQ ID NO:25; each start position isspecified, the length of peptide is 10 amino acids, and the end positionfor each peptide is the start position plus nine. 2 RALDGEESLL 14.400 9SLLSKYNSNL 6.000 1 QRALDGEESL 0.400 5 DGEESLLSKY 0.238 8 ESLLSKYNSN0.180 3 ALDGEESLLS 0.100 6 GEESLLSKYN 0.018 7 EESLLSKYNS 0.010 4LDGEESLLSK 0.001

TABLE XVIII Start Subsequence Score V1-HLA-B7-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 39 YPMRILYDL 240.000 265TPSPIIQQL 80.000 114 NPPQAVNLL 80.000 95 SPQLSDFGL 80.000 286 VPTFCTPGL80.000 390 AVPPSKRFL 60.000 10 KLRSLASTL 40.000 163 SPRVKKNSV 40.000 367IPEDILQLL 24.000 304 ALVSTNYPL 12.000 250 EAIDTESRL 12.000 80 DPPVASSCI8.000 217 TPKLEHFGI 8.000 200 VLKTPKCAL 6.000 364 VTKIPEDIL 6.000 140KVLMEKNSM 5.000 189 VTPPTKQSL 4.000 3 PIRSFCGKL 4.000 314 KTNSSSNDL4.000 374 LLSKYNSNL 4.000 224 GISEYTMCL 4.000 190 TPPTKQSLV 4.000 119VNLLDKARL 4.000 366 KIPEDILQL 4.000 46 DLHSEVQTL 4.000 348 TISSYENLL4.000 21 ETARLQRAL 4.000 277 DAEYTNSPL 3.600 92 SPRSPQLSD 3.000 283SPLVPTFCT 3.000 185 EPVIVTPPT 2.000 261 NVFATPSPI 2.000 296 IPSTKNSIA2.000 363 EVTKIPEDI 2.000 291 TPGLKIPST 2.000 232 LNEDYTMGL 1.200 17TLDCETARL 1.200 333 NSDTCFENL 1.200 50 EVQTLKDDV 1.000 135 FIKATKVLM1.000 230 MCLNEDYTM 1.000 202 KTPKCALKM 1.000 383 ATPIAIKAV 0.600 343DPSSPTISS 0.600 112 LPNPPQAVN 0.600 322 LEVEDRTSL 0.600 110 QVLPNPPQA0.500 199 KVLKTPKCA 0.500 22 TARLQRALD 0.450 113 PNPPQAVNL 0.400 70QKTDVKDDL 0.400 297 PSTKNSIAL 0.400 145 KNSMDIMKI 0.400 169 NSVHEQEAI0.400 347 PTISSYENL 0.400 90 GKSPRSPQL 0.400 134 DFIKATKVL 0.400 310YPLSKTNSS 0.400 346 SPTISSYEN 0.400 378 YNSNLATPI 0.400 6 SFCGKLRSL0.400 193 TKQSLVKVL 0.400 180 DNYKEEPVI 0.400 295 KIPSTKNSI 0.400 244RNNKSEEAI 0.400 104 ERYIVSQVL 0.400 128 ENQEGIDFI 0.400 380 SNLATPIAI0.400 52 QTLKDDVNI 0.400 323 EVEDRTSLV 0.300 117 QAVNLLDKA 0.300 15ASTLDCETA 0.300 33 ESDFEDYPM 0.300 382 LATPIAIKA 0.300 242 NARNNKSEE0.300 207 ALKMDDFEC 0.300 111 VLPNPPQAV 0.300 391 VPPSKRFLK 0.300 124KARLENQEG 0.300 358 TPTPPEVTK 0.300 14 LASTLDCET 0.300 384 TPIAIKAVP0.200 360 TPPEVTKIP 0.200 2 DPIRSFCGK 0.200 298 STKNSIALV 0.200 152KIREYFQKY 0.200 43 ILYDLHSEV 0.200 203 TPKCALKMD 0.200 316 NSSSNDLEV0.200 267 SPIIQQLEK 0.200 103 LERYIVSQV 0.200 255 ESRLNDNVF 0.200 36FEDYPMRIL 0.180 355 LLRTPTPPE 0.150 280 YTNSPLVPT 0.150 57 DVNIPELSN0.150 340 NLTDPSSPT 0.150 7 FCGKLRSLA 0.150 118 AVNLLDKAR 0.150 307STNYPLSKT 0.150 V5-HLA-B7-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 9 EKSPRSPQL 0.400 3 ASSCISEKS 0.060 2 VASSCISEK0.030 6 CISEKSPRS 0.020 5 SCISEKSPR 0.010 4 SSCISEKSP 0.010 1 PVASSCISE0.005 7 ISEKSPRSP 0.003 8 SEKSPRSPQ 0.002 V6-HLA-B7-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 5 EAIDAESRL 12.000 9 AESRLNDNV0.060 6 AIDAESRLN 0.018 8 DAESRLNDN 0.018 1 NKSEEAIDA 0.010 2 KSEEAIDAE0.003 7 IDAESRLND 0.002 4 EEAIDAESR 0.001 3 SEEAIDAES 0.001V10-HLA-B7-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 14 YPTQKLNKM 20.000 11 QWIYPTQKL 0.600 5 DILQKFQWI 0.400 2IPEDILQKF 0.120 12 WIYPTQKLN 0.020 6 ILQKFQWIY 0.020 8 QKFQWIYPT 0.010 7LQKFQWIYP 0.010 10 FQWIYPTQK 0.010 1 KIPEDILQK 0.010 4 EDILQKFQW 0.00213 IYPTQKLNK 0.001 15 PTQKLNKMR 0.001 9 KFQWIYPTQ 0.001 3 PEDILQKFQ0.000 V12-HLA-B7-9mers-193P1E1B Each peptide is a portion of SEQ IDNO:25; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 1 RALDGEESL 12.000 2 ALDGEESLL 3.600 7 ESLLSKYNS 0.020 8SLLSKYNSN 0.020 4 DGEESLLSK 0.003 3 LDGEESLLS 0.002 6 EESLLSKYN 0.002 5GEESLLSKY 0.001

TABLE XIX Start Subsequence Score V1-HLA-B7-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 296 IPSTKNSIAL 80.000 2DPIRSFCGKL 80.000 112 LPNPPQAVNL 80.000 346 SPTISSYENL 80.000 118AVNLLDKARL 60.000 363 EVTKIPEDIL 30.000 199 KVLKTPKCAL 30.000 188IVTPPTKQSL 20.000 285 LVPTFCTPGL 20.000 303 IALVSTNYPL 12.000 389KAVPPSKRFL 12.000 264 ATPSPIIQQL 12.000 124 KARLENQEGI 12.000 358TPTPPEVTKI 8.000 323 EVEDRTSLVL 6.000 231 CLNEDYTMGL 4.000 366KIPEDILQLL 4.000 103 LERYIVSQVL 4.000 69 FQKTDVKDDL 4.000 92 SPRSPQLSDF4.000 94 RSPQLSDFGL 4.000 5 RSFCGKLRSL 4.000 223 FGISEYTMCL 4.000 332LNSDTCFENL 4.000 16 STLDCETARL 4.000 89 SGKSPRSPQL 4.000 373 QLLSKYNSNL4.000 261 NVFATPSPII 3.000 242 NARNNKSEEA 3.000 355 LLRTPTPPEV 2.000 163SPRVKKNSVH 2.000 321 DLEVEDRTSL 1.800 110 QVLPNPPQAV 1.500 141VLMEKNSMDI 1.200 255 ESRLNDNVFA 1.000 229 TMCLNEDYTM 1.000 207ALKMDDFECV 0.600 382 LATPIAIKAV 0.600 39 YPMRILYDLH 0.600 57 DVNIPELSNC0.500 197 LVKVLKTPKC 0.500 133 IDFIKATKVL 0.400 379 NSNLATPIAI 0.400 217TPKLEHFGIS 0.400 191 PPTKQSLVKV 0.400 365 TKIPEDILQL 0.400 343DPSSPTISSY 0.400 9 GKLRSLASTL 0.400 168 KNSVHEQEAI 0.400 276 SDAEYTNSPL0.400 38 DYPMRILYDL 0.400 216 VTPKLEHFGI 0.400 211 DDFECVTPKL 0.400 45YDLHSEVQTL 0.400 51 VQTLKDDVNI 0.400 20 CETARLQRAL 0.400 260 DNVFATPSPI0.400 192 PTKQSLVKVL 0.400 313 SKTNSSSNDL 0.400 340 NLTDPSSPTI 0.400 267SPIIQQLEKS 0.400 113 PNPPQAVNLL 0.400 249 EEAIDTESRL 0.400 80 DPPVASSCIS0.400 347 PTISSYENLL 0.400 310 YPLSKTNSSS 0.400 228 YTMCLNEDYT 0.300 14LASTLDCETA 0.300 22 TARLQRALDG 0.300 142 LMEKNSMDIM 0.300 206 CALKMDDFEC0.300 390 AVPPSKRFLK 0.225 283 SPLVPTFCTP 0.200 25 LQRALDGEES 0.200 291TPGLKIPSTK 0.200 265 TPSPIIQQLE 0.200 185 EPVIVTPPTK 0.200 180DNYKEEPVIV 0.200 203 TPKCALKMDD 0.200 286 VPTFCTPGLK 0.200 403NIRDVSNKEN 0.200 391 VPPSKRFLKH 0.200 189 VTPPTKQSLV 0.200 114NPPQAVNLLD 0.200 384 TPIAIKAVPP 0.200 42 RILYDLHSEV 0.200 95 SPQLSDFGLE0.200 315 TNSSSNDLEV 0.200 190 TPPTKQSLVK 0.200 157 FQKYGYSPRV 0.200 360TPPEVTKIPE 0.200 162 YSPRVKKNSV 0.200 35 DFEDYPMRIL 0.180 277 DAEYTNSPLV0.180 306 VSTNYPLSKT 0.150 339 ENLTDPSSPT 0.150 282 NSPLVPTFCT 0.150 60IPELSNCENF 0.120 243 ARNNKSEEAI 0.120 367 IPEDILQLLS 0.120V5-HLA-B7-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:11;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 9 SEKSPRSPQL 0.400 3 VASSCISEKS 0.060 4 ASSCISEKSP 0.030 6SCISEKSPRS 0.020 1 PPVASSCISE 0.020 5 SSCISEKSPR 0.010 7 CISEKSPRSP0.010 8 ISEKSPRSPQ 0.007 2 PVASSCISEK 0.005 10 EKSPRSPQLS 0.002V6-HLA-B7-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 5 EEAIDAESRL 0.400 9 DAESRLNDNV 0.180 1 NNKSEEAIDA 0.100 6EAIDAESRLN 0.060 7 AIDAESRLND 0.013 10 AESRLNDNVF 0.006 3 KSEEAIDAES0.006 8 IDAESRLNDN 0.002 2 NKSEEAIDAE 0.001 4 SEEAIDAESR 0.000V10-HLA-B7-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 11 FQWIYPTQKL 6.000 15 YPTQKLNKMR 0.200 14 IYPTQKLNKM 0.100 8LQKFQWIYPT 0.100 3 IPEDILQKFQ 0.060 5 EDILQKFQWI 0.040 6 DILQKFQWIY0.020 2 KIPEDILQKF 0.020 13 WIYPTQKLNK 0.010 7 ILQKFQWIYP 0.010 12QWIYPTQKLN 0.002 9 QKFQWIYPTQ 0.001 10 KFQWIYPTQK 0.001 1 TKIPEDILQK0.001 4 PEDILQKFQW 0.000 V12-HLA-B7-10mers-193P1E1B Each peptide is aportion of SEQ ID NO:25; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. 2 RALDGEESLL 12.000 9 SLLSKYNSNL 4.000 1QRALDGEESL 0.400 8 ESLLSKYNSN 0.020 3 ALDGEESLLS 0.018 5 DGEESLLSKY0.006 7 EESLLSKYNS 0.002 4 LDGEESLLSK 0.001 6 GEESLLSKYN 0.001

TABLE XX Start Subsequence Score V1-HLA-B3501-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 217 TPKLEHFGI 36.000 152KIREYFQKY 24.000 39 YPMRILYDL 20.000 95 SPQLSDFGL 20.000 114 NPPQAVNLL20.000 286 VPTFCTPGL 20.000 265 TPSPIIQQL 20.000 255 ESRLNDNVF 15.000163 SPRVKKNSV 12.000 367 IPEDILQLL 12.000 80 DPPVASSCI 8.000 250EAIDTESRL 6.000 366 KIPEDILQL 6.000 140 KVLMEKNSM 6.000 135 FIKATKVLM6.000 389 KAVPPSKRF 6.000 10 KLRSLASTL 6.000 33 ESDFEDYPM 4.500 202KTPKCALKM 4.000 190 TPPTKQSLV 4.000 230 MCLNED YTM 3.000 364 VTKIPEDIL3.000 98 LSDFGLERY 3.000 200 VLKTPKCAL 3.000 169 NSVHEQEAI 3.000 302SIALVSTNY 2.000 283 SPLVPTFCT 2.000 296 IPSTKNSIA 2.000 112 LPNPPQAVN2.000 310 YPLSKTNSS 2.000 370 DILQLLSKY 2.000 224 GISEYTMCL 2.000 185EPVIVTPPT 2.000 228 YTMCLNEDY 2.000 314 KTNSSSNDL 2.000 346 SPTISSYEN2.000 205 KCALKMDDF 2.000 291 TPGLKIPST 2.000 343 DPSSPTISS 2.000 312LSKTNSSSN 1.500 46 DLHSEVQTL 1.500 119 VNLLDKARL 1.500 333 NSDTCFENL1.500 375 LSKYNSNLA 1.500 145 KNSMDIMKI 1.200 5 RSFCGKLRS 1.000 215CVTPKLEHF 1.000 390 AVPPSKRFL 1.000 304 ALVSTNYPL 1.000 189 VTPPTKQSL1.000 350 SSYENLLRT 1.000 149 DIMKIREYF 1.000 374 LLSKYNSNL 1.000 316NSSSNDLEV 1.000 330 LVLNSDTCF 1.000 344 PSSPTISSY 1.000 348 TISSYENLL1.000 91 KSPRSPQLS 1.000 12 RSLASTLDC 1.000 281 TNSPLVPTF 1.000 21ETARLQRAL 1.000 277 DAEYTNSPL 0.900 244 RNNKSEEAI 0.800 128 ENQEGIDFI0.800 295 KIPSTKNSI 0.800 15 ASTLDCETA 0.750 272 QLEKSDAEY 0.600 60IPELSNCEN 0.600 143 MEKNSMDIM 0.600 180 DNYKEEPVI 0.600 52 QTLKDDVNI0.600 298 STKNSIALV 0.600 92 SPRSPQLSD 0.600 232 LNEDYTMGL 0.600 203TPKCALKMD 0.600 379 NSNLATPIA 0.500 162 YSPRVKKNS 0.500 328 TSLVLNSDT0.500 297 PSTKNSIAL 0.500 301 NSIALVSTN 0.500 282 NSPLVPTFC 0.500 195QSLVKVLKT 0.500 84 ASSCISGKS 0.500 17 TLDCETARL 0.450 28 ALDGEESDF 0.450207 ALKMDDFEC 0.450 275 KSDAEYTNS 0.450 380 SNLATPIAI 0.400 261NVFATPSPI 0.400 154 REYFQKYGY 0.400 363 EVTKIPEDI 0.400 360 TPPEVTKIP0.400 43 ILYDLHSEV 0.400 378 YNSNLATPI 0.400 257 RLNDNVFAT 0.400 386IAIKAVPPS 0.300 8 CGKLRSLAS 0.300 117 QAVNLLDKA 0.300 14 LASTLDCET 0.3003 PIRSFCGKL 0.300 V5-HLA-B3501-9mers-193P1E1B Each peptide is a portionof SEQ ID NO:11; each start position is specified, the length of peptideis 9 amino acids, and the end position for each peptide is the startposition plus eight. 3 ASSCISEKS 0.500 6 CISEKSPRS 0.200 9 EKSPRSPQL0.100 4 SSCISEKSP 0.500 2 VASSCISEK 0.030 7 ISEKSPRSP 0.015 5 SCISEKSPR0.015 8 SEKSPRSPQ 0.003 1 PVASSCISE 0.001 V6-HLA-B3501-9mers-193P1E1BEach peptide is a portion of SEQ ID NO:13; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plus eight. 5 EAIDAESRL 6.000 8DAESRLNDN 0.090 2 KSEEAIDAE 0.060 6 AIDAESRLN 0.045 1 NKSEEAIDA 0.030 9AESRLNDNV 0.020 3 SEEAIDAES 0.003 7 IDAESRLND 0.002 4 EEAIDAESR 0.002V10-HLA-B3501-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 14 YPTQKLNKM 40.000 2 IPEDILQKF 12.000 6 ILQKFQWIY 2.000 5DILQKFQWI 0.400 11 QWIYPTQKL 0.100 12 WIYPTQKLN 0.100 1 KIPEDILQK 0.0604 EDILQKFQW 0.050 7 LQKFQWIYP 0.030 10 FQWIYPTQK 0.010 8 QKFQWIYPT 0.0109 KFQWIYPTQ 0.002 13 IYPTQKLNK 0.001 15 PTQKLNKMR 0.001 3 PEDILQKFQ0.000 V12-HLA-B3501-9mers-193P1E1B Each peptide is a portion of SEQ IDNO:25; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 1 RALDGEESL 12.000 7 ESLLSKYNS 0.500 2 ALDGEESLL 0.450 8SLLSKYNSN 0.100 5 GEESLLSKY 0.060 3 LDGEESLLS 0.030 6 EESLLSKYN 0.010 4DGEESLLSK 0.006

TABLE XXI Start Subsequence Score V1-HLA-B3501-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 92 SPRSPQLSDF 60.000 343DPSSPTISSY 40.000 346 SPTISSYENL 20.000 2 DPIRSFCGKL 20.000 296IPSTKNSIAL 20.000 112 LPNPPQAVNL 20.000 27 RALDGEESDF 18.000 358TPTPPEVTKI 12.000 301 NSIALVSTNY 10.000 94 RSPQLSDFGL 10.000 5RSFCGKLRSL 10.000 124 KARLENQEGI 7.200 389 KAVPPSKRFL 6.000 217TPKLEHFGIS 6.000 60 IPELSNCENF 6.000 271 QQLEKSDAEY 4.000 366 KIPEDILQLL4.000 97 QLSDFGLERY 4.000 303 IALVSTNYPL 3.000 89 SGKSPRSPQL 3.000 69FQKTDVKDDL 3.000 229 TMCLNEDYTM 3.000 16 STLDCETARL 3.000 255 ESRLNDNVFA2.250 310 YPLSKTNSSS 2.000 199 KVLKTPKCAL 2.000 231 CLNEDYTMGL 2.000 332LNSDTCFENL 2.000 379 NSNLATPIAI 2.000 80 DPPVASSCIS 2.000 267 SPIIQQLEKS2.000 30 DGEESDFEDY 1.800 375 LSKYNSNLAT 1.500 118 AVNLLDKARL 1.500 168KNSVHEQEAI 1.200 367 IPEDILQLLS 1.200 219 KLEHFGISEY 1.200 188IVTPPTKQSL 1.000 280 YTNSPLVPTF 1.000 162 YSPRVKKNSV 1.000 329SLVLNSDTCF 1.000 363 EVTKIPEDIL 1.000 264 ATPSPIIQQL 1.000 223FGISEYTMCL 1.000 373 QLLSKYNSNL 1.000 285 LVPTFCTPGL 1.000 214ECVTPKLEHF 1.000 126 RLENQEGIDF 0.900 207 ALKMDDFECV 0.900 242NARNNKSEEA 0.900 250 EAIDTESRLN 0.900 340 NLTDPSSPTI 0.800 141VLMEKNSMDI 0.800 216 VTPKLEHFGI 0.600 323 EVEDRTSLVL 0.600 147SMDIMKIREY 0.600 382 LATPIAIKAV 0.600 32 EESDFEDYPM 0.600 157 FQKYGYSPRV0.600 203 TPKCALKMDD 0.600 51 VQTLKDDVNI 0.600 142 LMEKNSMDIM 0.600 137KATKVLMEKN 0.600 403 NIRDVSNKEN 0.600 247 KSEEAIDTES 0.600 98 LSDFGLERYI0.600 173 EQEAINSDNY 0.600 355 LLRTPTPPEV 0.600 163 SPRVKKNSVH 0.600 306VSTNYPLSKT 0.500 282 NSPLVPTFCT 0.500 345 SSPTISSYEN 0.500 169NSVHEQEAIN 0.500 328 TSLVLNSDTC 0.500 349 ISSYENLLRT 0.500 63 LSNCENFQKT0.500 25 LQRALDGEES 0.450 245 NNKSEEAIDT 0.450 14 LASTLDCETA 0.450 206CALKMDDFEC 0.450 321 DLEVEDRTSL 0.450 260 DNVFATPSPI 0.400 261NVFATPSPII 0.400 42 RILYDLHSEV 0.400 191 PPTKQSLVKV 0.400 360 TPPEVTKIPE0.400 138 ATKVLMEKNS 0.300 298 STKNSIALVS 0.300 180 DNYKEEPVIV 0.300 192PTKQSLVKVL 0.300 170 SVHEQEAINS 0.300 197 LVKVLKTPKC 0.300 239GLKNARNNKS 0.300 8 CGKLRSLAST 0.300 139 TKVLMEKNSM 0.300 103 LERYIVSQVL0.300 178 NSDNYKEEPV 0.300 83 VASSCISGKS 0.300 95 SPQLSDFGLE 0.300 293GLKIPSTKNS 0.300 V5-HLA-B3501-10mers-193P1E1B Each peptide is a portionof SEQ ID NO:11; each start position is specified, the length of peptideis 10 amino acids, and the end position for each peptide is the startposition plus nine. 9 SEKSPRSPQL 0.300 3 VASSCISEKS 0.300 6 SCISEKSPRS0.100 5 SSCISEKSPR 0.075 4 ASSCISEKSP 0.050 7 CISEKSPRSP 0.020 1PPVASSCISE 0.020 8 ISEKSPRSPQ 0.015 10 EKSPRSPQLS 0.010 2 PVASSCISEK0.001 V6-HLA-B3501-10mers-193P1E1B Each peptide is a portion of SEQ IDNO:13; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. 6 EAIDAESRLN 0.900 3 KSEEAIDAES 0.600 1 NNKSEEAIDA 0.450 9DAESRLNDNV 0.180 5 EEAIDAESRL 0.100 10 AESRLNDNVF 0.100 8 IDAESRLNDN0.020 7 AIDAESRLND 0.003 2 NKSEEAIDAE 0.002 4 SEEAIDAESR 0.000V10-HLA-B3501-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 2 KIPEDILQKF 4.000 6 DILQKFQWIY 2.000 11 FQWIYPTQKL 1.000 8LQKFQWIYPT 0.300 15 YPTQKLNKMR 0.200 14 IYPTQKLNKM 0.200 3 IPEDILQKFQ0.120 5 EDILQKFQWI 0.040 7 ILQKFQWIYP 0.010 12 QWIYPTQKLN 0.010 13WIYPTQKLNK 0.010 10 KFQWIYPTQK 0.002 4 PEDILQKFQW 0.002 1 TKIPEDILQK0.002 9 QKFQWIYPTQ 0.001 V12-HLA-B3501-10mers-193P1E1B Each peptide is aportion of SEQ ID NO:25; each start position is specified, the length ofpeptide is 10 amino acids, and the end position for each peptide is thestart position plus nine. 2 RALDGEESLL 18.000 5 DGEESLLSKY 1.200 9SLLSKYNSNL 1.000 8 ESLLSKYNSN 0.500 1 QRALDGEESL 0.100 3 ALDGEESLLS0.045 7 EESLLSKYNS 0.010 6 GEESLLSKYN 0.003 4 LDGEESLLSK 0.002

TABLE XII V1-HLA-A1-9mers-193P1E1B Each peptide is a portion of SEQ IDNO: 3; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. Pos 123456789 score  98 LSDFGLERY 31  31 GEESDFEDY 28  37EDYPMRILY 26 272 QLEKSDAEY 26  48 HSEVQTLKD 24 228 YTMCLNEDY 24 344PSSPTISSY 23 152 KIREYFQKY 21  78 LSDPPVASS 20 341 LTDPSSPTI 20 182YKEEPVIVT 19 302 SIALVSTNY 19  71 KTDVKDDLS 18 121 LLDKARLEN 18 324VEDRTSLVL 18 368 PEDILQLLS 18  19 DCETARLQR 17  54 LKDDVNIPE 17 154REYFQKYGY 17 220 LEHFGISEY 17 333 NSDTCFENL 17 370 DILQLLSKY 17 147SMDIMKIRE 16 183 KEEPVIVTP 16 219 KLEHFGISE 16 225 ISEYTMCLN 16 253DTESRLNDN 16 148 MDIMKIREY 15 171 VHEQEAINS 15 174 QEAINSDNY 15 178NSDNYKEEP 15 258 LNDNVFATP 15

TABLE XXII Pos 123456789 score V5-HLA-A1-9mers-193P1E1B Each peptide isa portion of SEQ ID NO: 11; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight.  7 ISEKSPRSP 14  3 ASSCISEKS  7  4SSCISEKSP  6 V6-HLA-A1-9mers-193P1E1B Each peptide is a portion of SEQID NO: 13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight.  2 KSEEAIDAE 14  6 AIDAESRLN 13  3 SEEAIDAES 12  8 DAESRLNDN10  7 IDAESRLND  7 V10-HLA-A1-9mers-193P1E1B Each peptide is a portionof SEQ ID NO: 21; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight.  6 ILQKFQWIY 16  2 IPEDILQKF 12  3 PEDILQKFQ10 13 IYPTQKLNK  8 15 PTQKLNKMR  8 V12-HLA-A1-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO: 25; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight.  5 GEESLLSKY 27  4 DGEESLLSK16  2 ALDGEESLL 15

TABLE XXIII Pos 123456789 score V1-HLA-A0201-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 43 ILYDLHSEV 27 366 KIPEDILQL26 46 DLHSEVQTL 25 10 KLRSLASTL 24 17 TLDCETARL 24 224 GISEYTMCL 24 111VLPNPPQAV 23 304 ALVSTNYPL 23 374 LLSKYNSNL 23 200 VLKTPKCAL 22 6SFCGKLRSL 21 257 RLNDNVFAT 21 298 STKNSIALV 21 295 KIPSTKNSI 20 348TISSYENLL 20 383 ATPIAIKAV 20 102 GLERYIVSQ 19 189 VTPPTKQSL 19 341LTDPSSPTI 19 381 NLATPIAIK 19 3 PIRSFCGKL 18 13 SLASTLDCE 18 39YPMRILYDL 18 55 KDDVNIPEL 18 192 PTKQSLVKV 18 280 YTNSPLVPT 18 390AVPPSKRFL 18 142 LMEKNSMDI 17 196 SLVKVLKTP 17 307 STNYPLSKT 17 314KTNSSSNDL 17 359 PTPPEVTKI 17 42 RILYDLHSE 16 52 QTLKDDVNI 16 133IDFIKATKV 16 163 SPRVKKNSV 16 232 LNEDYTMGL 16 265 TPSPIIQQL 16 322LEVEDRTSL 16 355 LLRTPTPPE 16 356 LRTPTPPEV 16 367 IPEDILQLL 16 370DILQLLSKY 16 373 QLLSKYNSN 16 9 GKLRSLAST 15 21 ETARLQRAL 15 24RLQRALDGE 15 53 TLKDDVNIP 15 97 QLSDFGLER 15 103 LERYIVSQV 15 106YIVSQVLPN 15 114 NPPQAVNLL 15 117 QAVNLLDKA 15 119 VNLLDKARL 15 121LLDKARLEN 15 125 ARLENQEGI 15 141 VLMEKNSMD 15 145 KNSMDIMKI 15 158QKYGYSPRV 15 209 KMDDFECVT 15 340 NLTDPSSPT 15 364 VTKIPEDIL 15 399KHGQNIRDV 15 28 ALDGEESDF 14 77 DLSDPPVAS 14 90 GKSPRSPQL 14 99SDFGLERYI 14 120 NLLDKARLE 14 135 FIKATKVLM 14 140 KVLMEKNSM 14 152KIREYFQKY 14 176 AINSDNYKE 14 193 TKQSLVKVL 14 195 QSLVKVLKT 14 208LKMDDFECV 14 250 EAIDTESRL 14 278 AEYTNSPLV 14 329 SLVLNSDTC 14 331VLNSDTCFE 14 350 SSYENLLRT 14 14 LASTLDCET 13 78 LSDPPVASS 13 95SPQLSDFGL 13 110 QVLPNPPQA 13 128 ENQEGIDFI 13 179 SDNYKEEPV 13 181NYKEEPVIV 13 207 ALKMDDFEC 13 212 DFECVTPKL 13 219 KLEHFGISE 13 231CLNEDYTMG 13 261 NVFATPSPI 13 262 VFATPSPII 13 268 PIIQQLEKS 13 272QLEKSDAEY 13 286 VPTFCTPGL 13 293 GLKIPSTKN 13 300 KNSIALVST 13 316NSSSNDLEV 13 323 EVEDRTSLV 13 347 PTISSYENL 13 380 SNLATPIAI 13 382LATPIAIKA 13 386 IAIKAVPPS 13 397 FLKHGQNIR 13 50 EVQTLKDDV 12 59NIPELSNCE 12 75 KDDLSDPPV 12 87 CISGKSPRS 12 132 GIDFIKATK 12 134DFIKATKVL 12 182 YKEEPVIVT 12 187 VIVTPPTKQ 12 202 KTPKCALKM 12 229TMCLNEDYT 12 236 YTMGLKNAR 12 269 IIQQLEKSD 12 276 SDAEYTNSP 12 288TFCTPGLKI 12 291 TPGLKIPST 12 302 SIALVSTNY 12 324 VEDRTSLVL 12 354NLLRTPTPP 12 371 ILQLLSKYN 12 387 AIKAVPPSK 12 403 NIRDVSNKE 12V5-HLA-A0201-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:11;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 6 CISEKSPRS 12 2 VASSCISEK 11 9 EKSPRSPQL 10 1 PVASSCISE 5V6-HLA-A0201-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 EAIDAESRL 14 9 AESRLNDNV 13 6 AIDAESRLN 10 2 KSEEAIDAE 7 7IDAESRLND 7 8 DAESRLNDN 7 1 NKSEEAIDA 6 V10-HLA-A0201-9mers-193P1E1BEach peptide is a portion is SEQ ID NO:21; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plus eight. 5 DILQKFQWI 18 1KIPEDILQK 16 11 QWIYPTQKL 16 6 ILQKFQWIY 13 14 YPTQKLNKM 13 8 QKFQWIYPT10 12 WIYPTQKLN 10 2 IPEDILQKF 8 V12-HLA-A0201-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:25; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 2 ALDGEESLL 24 1 RALDGEESL 218 SLLSKYNSN 18

TABLE XXIV Pos 123456789 score V1-HLA-A0203-9mers-193P1E1BNoResultsFound. V5-HLA-A0203-9mers-193P1E1B NoResultsFound.V6-HLA-A0203-9mers-193P1E1B NoResultsFound. V10-HLA-A0203-9mers-193P1E1BNoResultsFound. V12-HLA-A0203-9mers-193P1E1B NoResultsFound.

TABLE XXV Pos 123456789 score V1-HLA-A3-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:3; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 186 PVIVTPPTK 28 387 AIKAVPPSK 28 10KLRSLASTL 26 132 GIDFIKATK 25 381 NLATPIAIK 24 97 QLSDFGLER 23 239GLKNARNNK 23 28 ALDGEESDF 22 197 LVKVLKTPK 22 110 QVLPNPPQA 21 152KIREYFQKY 21 272 QLEKSDAEY 21 358 TPTPPEVTK 21 402 QNIRDVSNK 21 43ILYDLHSEV 20 292 PGLKIPSTK 20 102 GLERYIVSQ 19 118 AVNLLDKAR 19 151MKIREYFQK 19 160 YGYSPRVKK 19 165 RVKKNSVHE 19 194 KQSLVKVLK 19 302SIALVSTNY 19 369 EDILQLLSK 19 370 DILQLLSKY 19 159 KYGYSPRVK 18 188IVTPPTKQS 18 215 CVTPKLEHF 18 219 KLEHFGISE 18 267 SPIIQQLEK 18 330LVLNSDTCF 18 366 KIPEDILQL 18 385 PIAIKAVPP 18 24 RLQRALDGE 17 77DLSDPPVAS 17 120 NLLDKARLE 17 140 KVLMEKNSM 17 191 PPTKQSLVK 17 201LKTPKCALK 17 284 PLVPTFCTP 17 306 VSTNYPLSK 17 354 NLLRTPTPP 17 373QLLSKYNSN 17 397 FLKHGQNIR 17 2 DPIRSFCGK 16 42 RILYDLHSE 16 57DVNIPELSN 16 126 RLENQEGID 16 141 VLMEKNSMD 16 196 SLVKVLKTP 16 199KVLKTPKCA 16 257 RLNDNVFAT 16 261 NVFATPSPI 16 323 EVEDRTSLV 16 329SLVLNSDTC 16 390 AVPPSKRFL 16 67 ENFQKTDVK 15 73 DVKDDLSDP 15 135FIKATKVLM 15 137 KATKVLMEK 15 170 SVHEQEAIN 15 183 KEEPVIVTP 15 311PLSKTNSSS 15 37 EDYPMRILY 14 46 DLHSEVQTL 14 116 PQAVNLLDK 14 121LLDKARLEN 14 154 REYFQKYGY 14 175 EAINSDNYK 14 207 ALKMDDFEC 14 321DLEVEDRTS 14 340 NLTDPSSPT 14 344 PSSPTISSY 14 17 TLDCETARL 13 23ARLQRALDG 13 47 LHSEVQTLK 13 63 LSNCENFQK 13 79 SDPPVASSC 13 82PVASSCISG 13 83 VASSCISGK 13 86 SCISGKSPR 13 107 IVSQVLPNP 13 111VLPNPPQAV 13 149 DIMKIREYF 13 231 CLNEDYTMG 13 293 GLKIPSTKN 13 295KIPSTKNSI 13 304 ALVSTNYPL 13 355 LLRTPTPPE 13 371 ILQLLSKYN 13 374LLSKYNSNL 13 403 NIRDVSNKE 13 V5-HLA-A3-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:11; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 1 PVASSCISE 13 2 VASSCISEK 13 5 SCISEKSPR 116 CISEKSPRS 10 9 EKSPRSPQL 8 8 SEKSPRSPQ 6 V6-HLA-A3-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 6 AIDAESRLN 13 4 EEAIDAESR 117 IDAESRLND 8 5 EAIDAESRL 7 3 SEEAIDAES 6 9 AESRLNDNV 6V10-HLA-A3-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 1 KIPEDILQK 28 6 ILQKFQWIY 18 10 FQWIYPTQK 16 12 WIYPTQKLN 15 13IYPTQKLNK 15 V12-HLA-A3-9mers-193P1E1B Each peptide is a portion of SEQID NO:25; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 2 ALDGEESLL 18 4 DGEESLLSK 16 8 SLLSKYNSN 16 1 RALDGEESL 115 GEESLLSKY 9

TABLE XXVI Pos 123456789 score V1-HLA-A26-9mers-193P1E1B Each peptide isa portion of SEQ ID NO:3; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight. 370 DILQLLSKY 29 21 ETARLQRAL 28 215CVTPKLEHF 25 73 DVKDDLSDP 24 250 EAIDTESRL 24 363 EVTKIPEDI 23 37EDYPMRILY 22 46 DLHSEVQTL 22 149 DIMKIREYF 22 323 EVEDRTSLV 22 50EVQTLKDDV 21 253 DTESRLNDN 21 347 PTISSYENL 21 57 DVNIPELSN 20 131EGIDFIKAT 20 134 DFIKATKVL 20 366 KIPEDILQL 20 369 EDILQLLSK 20 148MDIMKIREY 19 228 YTMCLNEDY 19 255 ESRLNDNVF 19 390 AVPPSKRFL 19 104ERYIVSQVL 18 189 VTPPTKQSL 18 211 DDFECVTPK 18 330 LVLNSDTCF 18 335DTCFENLTD 18 152 KIREYFQKY 17 212 DFECVTPKL 17 265 TPSPIIQQL 17 277DAEYTNSPL 17 314 KTNSSSNDL 17 344 PSSPTISSY 17 128 ENQEGIDFI 16 155EYFQKYGYS 16 184 EEPVIVTPP 16 221 EHFGISEYT 16 281 TNSPLVPTF 16 326DRTSLVLNS 16 364 VTKIPEDIL 16 3 PIRSFCGKL 15 6 SFCGKLRSL 15 67 ENFQKTDVK15 186 PVIVTPPTK 15 193 TKQSLVKVL 15 214 ECVTPKLEH 15 220 LEHFGISEY 15227 EYTMCLNED 15 261 NVFATPSPI 15 264 ATPSPIIQQ 15 302 SIALVSTNY 15 307STNYPLSKT 15 322 LEVEDRTSL 15 367 IPEDILQLL 15 39 YPMRILYDL 14 56DDVNIPELS 14 93 PRSPQLSDF 14 98 LSDFGLERY 14 106 YIVSQVLPN 14 107IVSQVLPNP 14 175 EAINSDNYK 14 185 EPVIVTPPT 14 224 GISEYTMCL 14 325EDRTSLVLN 14 348 TISSYENLL 14 359 PTPPEVTKI 14 V5-HLA-A26-9mers-193P1E1BEach peptide is a portion of SEQ ID NO:11; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plus eight. 9 EKSPRSPQL 20 1PVASSCISE 13 V6-HLA-A26-9mers-193P1E1B Each peptide is a portion of SEQID NO:13; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 5 EAIDAESRL 24 8 DAESRLNDN 13 4 EEAIDAESR 11V10-HLA-A26-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 2 IPEDILQKF 16 4 EDILQKFQW 15 5 DILQKFQWI 13 11 QWIYPTQKL 13 1KIPEDILQK 12 6 ILQKFQWIY 10 15 PTQKLNKMR 10 8 QKFQWIYPT 8 14 YPTQKLNKM 7V12-HLA-A26-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 GEESLLSKY 18 4 DGEESLLSK 16 6 EESLLSKYN 11 1 RALDGEESL 10 7ESLLSKYNS 10 2 ALDGEESLL 9

TABLE XXVII Pos 123456789 score V1-HLA-B0702-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 265 TPSPIIQQL 23 286 VPTFCTPGL22 39 YPMRILYDL 21 114 NPPQAVNLL 21 367 IPEDILQLL 21 95 SPQLSDFGL 20 185EPVIVTPPT 20 283 SPLVPTFCT 20 296 IPSTKNSIA 19 163 SPRVKKNSV 18 291TPGLKIPST 18 92 SPRSPQLSD 17 80 DPPVASSCI 16 112 LPNPPQAVN 16 190TPPTKQSLV 16 217 TPKLEHFGI 16 343 DPSSPTISS 16 358 TPTPPEVTK 16 392PPSKRFLKH 16 36 FEDYPMRIL 14 90 GKSPRSPQL 14 191 PPTKQSLVK 14 200VLKTPKCAL 14 324 VEDRTSLVL 14 390 AVPPSKRFL 14 10 KLRSLASTL 13 17TLDCETARL 13 21 ETARLQRAL 13 55 KDDVNIPEL 13 113 PNPPQAVNL 13 115PPQAVNLLD 13 134 DFIKATKVL 13 224 GISEYTMCL 13 304 ALVSTNYPL 13 364VTKIPEDIL 13 366 KIPEDILQL 13 374 LLSKYNSNL 13 384 TPIAIKAVP 13 3PIRSFCGKL 12 6 SFCGKLRSL 12 104 ERYIVSQVL 12 193 TKQSLVKVL 12 212DFECVTPKL 12 267 SPIIQQLEK 12 280 YTNSPLVPT 12 288 TFCTPGLKI 12 297PSTKNSIAL 12 322 LEVEDRTSL 12 333 NSDTCFENL 12 348 TISSYENLL 12 2DPIRSFCGK 11 28 ALDGEESDF 11 46 DLHSEVQTL 11 60 IPELSNCEN 11 81PPVASSCIS 11 119 VNLLDKARL 11 182 YKEEPVIVT 11 189 VTPPTKQSL 11 232LNEDYTMGL 11 250 EAIDTESRL 11 262 VFATPSPII 11 277 DAEYTNSPL 11 281TNSPLVPTF 11 300 KNSIALVST 11 310 YPLSKTNSS 11 314 KTNSSSNDL 11 357RTPTPPEVT 11 360 TPPEVTKIP 11 361 PPEVTKIPE 11 389 KAVPPSKRF 11 391VPPSKRFLK 11 V5-HLA-B0702-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 9 EKSPRSPQL 15 V6-HLA-B0702-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 5 EAIDAESRL 11 9 AESRLNDNV 101 NKSEEAIDA 8 7 IDAESRLND 5 V10-HLA-B0702-9mers-193P1E1B Each peptide isa portion of SEQ ID NO:21; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight. 2 IPEDILQKF 17 14 YPTQKLNKM 16 11QWIYPTQKL 14 V12-HLA-B0702-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:25; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 2 ALDGEESLL 15 1 RALDGEESL 11

TABLE XXVIII Pos 123456789 score V1-HLA-B08-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 200 VLKTPKCAL 28 163 SPRVKKNSV25 217 TPKLEHFGI 23 6 SFCGKLRSL 22 10 KLRSLASTL 22 364 VTKIPEDIL 21 3PIRSFCGKL 20 8 CGKLRSLAS 20 141 VLMEKNSMD 20 90 GKSPRSPQL 19 95SPQLSDFGL 19 150 IMKIREYFQ 19 122 LDKARLENQ 18 291 TPGLKIPST 18 296IPSTKNSIA 18 46 DLHSEVQTL 17 53 TLKDDVNIP 17 114 NPPQAVNLL 17 203TPKCALKMD 17 205 KCALKMDDF 17 207 ALKMDDFEC 17 224 GISEYTMCL 17 239GLKNARNNK 17 265 TPSPIIQQL 17 286 VPTFCTPGL 17 293 GLKIPSTKN 17 304ALVSTNYPL 17 366 KIPEDILQL 17 367 IPEDILQLL 17 374 LLSKYNSNL 17 391VPPSKRFLK 17 397 FLKHGQNIR 17 17 TLDCETARL 16 39 YPMRILYDL 16 120NLLDKARLE 16 135 FIKATKVLM 16 190 TPPTKQSLV 16 215 CVTPKLEHF 16 250EAIDTESRL 16 277 DAEYTNSPL 16 310 YPLSKTNSS 16 373 QLLSKYNSN 16 255ESRLNDNVF 15 385 PIAIKAVPP 15 87 CISGKSPRS 14 92 SPRSPQLSD 14 348TISSYENLL 14 387 AIKAVPPSK 14 392 PPSKRFLKH 14 21 ETARLQRAL 13 55KDDVNIPEL 13 69 FQKTDVKDD 13 80 DPPVASSCI 13 124 KARLENQEG 13 166VKKNSVHEQ 13 179 SDNYKEEPV 13 181 NYKEEPVIV 13 271 QQLEKSDAE 13 298STKNSIALV 13 V5-HLA-B08-9mers-193P1E1B Each peptide is a portion of SEQID NO:11; each start position is specified, the length of peptide is 9amino acids, and the end position for each peptide is the start positionplus eight. 9 EKSPRSPQL 20 6 CISEKSPRS 16 8 SEKSPRSPQ 12V6-HLA-B08-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 EAIDAESRL 16 8 DAESRLNDN 12 V10-HLA-B08-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:21; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 5 DILQKFQWI 20 14 YPTQKLNKM 162 IPEDILQKF 13 11 QWIYPTQKL 11 7 LQKFQWIYP 10 V12-HLA-B08-9mers-193P1E1BEach peptide is a portion of SEQ ID NO:25; each start position isspecified, the length of peptide is 9 amino acids, and the end positionfor each peptide is the start position plus eight. 8 SLLSKYNSN 18 2ALDGEESLL 16 1 RALDGEESL 14

TABLE XXIX Pos 123456789 score V1-HLA-B1510-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 21 ETARLQRAL 15 55 KDDVNIPEL15 90 GKSPRSPQL 15 265 TPSPIIQQL 15 390 AVPPSKRFL 15 399 KHGQNIRDV 15 36FEDYPMRIL 14 113 PNPPQAVNL 14 193 TKQSLVKVL 14 250 EAIDTESRL 14 367IPEDILQLL 14 6 SFCGKLRSL 13 17 TLDCETARL 13 104 ERYIVSQVL 13 119VNLLDKARL 13 134 DFIKATKVL 13 189 VTPPTKQSL 13 200 VLKTPKCAL 13 224GISEYTMCL 13 281 TNSPLVPTF 13 297 PSTKNSIAL 13 46 DLHSEVQTL 12 47LHSEVQTLK 12 70 QKTDVKDDL 12 114 NPPQAVNLL 12 171 VHEQEAINS 12 212DFECVTPKL 12 221 EHFGISEYT 12 232 LNEDYTMGL 12 322 LEVEDRTSL 12 324VEDRTSLVL 12 348 TISSYENLL 12 366 KIPEDILQL 12 374 LLSKYNSNL 12 10KLRSLASTL 11 39 YPMRILYDL 11 277 DAEYTNSPL 11 286 VPTFCTPGL 11 364VTKIPEDIL 11 389 KAVPPSKRF 11 3 PIRSFCGKL 10 95 SPQLSDFGL 10 255ESRLNDNVF 10 304 ALVSTNYPL 10 314 KTNSSSNDL 10 333 NSDTCFENL 10 347PTISSYENL 10 93 PRSPQLSDF 9 135 FIKATKVLM 9 149 DIMKIREYF 8 205KCALKMDDF 8 215 CVTPKLEHF 8 28 ALDGEESDF 7 33 ESDFEDYPM 7 61 PELSNCENF 777 DLSDPPVAS 7 127 LENQEGIDF 7 140 KVLMEKNSM 7 143 MEKNSMDIM 7 182YKEEPVIVT 7 183 KEEPVIVTP 7 202 KTPKCALKM 7 222 HFGISEYTM 7 230MCLNEDYTM 7 358 TPTPPEVTK 7 V5-HLA-B1510-9mers-193P1E1B Each peptide isa portion of SEQ ID NO:11; each start position is specified, the lengthof peptide is 9 amino acids, and the end position for each peptide isthe start position plus eight. 9 EKSPRSPQL 15 7 ISEKSPRSP 7V6-HLA-B1510-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 EAIDAESRL 14 V10-HLA-B1510-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:21; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 11 QWIYPTQKL 11 2 IPEDILQKF 10 14 YPTQKLNKM 8V12-HLA-B1510-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 1 RALDGEESL 12 2 ALDGEESLL 11

TABLE XXX Pos 123456789 score V1-HLA-B2705-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 104 ERYIVSQVL 25 164 PRVKKNSVH25 93 PRSPQLSDF 23 125 ARLENQEGI 22 4 IRSFCGKLR 21 137 KATKVLMEK 18 366KIPEDILQL 18 389 KAVPPSKRF 18 395 KRFLKHGQN 18 23 ARLQRALDG 17 55KDDVNIPEL 17 67 ENFQKTDVK 17 90 GKSPRSPQL 17 119 VNLLDKARL 17 132GIDFIKATK 17 211 DDFECVTPK 17 292 PGLKIPSTK 17 370 DILQLLSKY 17 10KLRSLASTL 16 47 LHSEVQTLK 16 86 SCISGKSPR 16 140 KVLMEKNSM 16 154REYFQKYGY 16 160 YGYSPRVKK 16 194 KQSLVKVLK 16 202 KTPKCALKM 16 239GLKNARNNK 16 265 TPSPIIQQL 16 267 SPIIQQLEK 16 330 LVLNSDTCF 16 369EDILQLLSK 16 374 LLSKYNSNL 16 396 RFLKHGQNI 16 402 QNIRDVSNK 16 6SFCGKLRSL 15 28 ALDGEESDF 15 34 SDFEDYPMR 15 41 MRILYDLHS 15 113PNPPQAVNL 15 134 DFIKATKVL 15 148 MDIMKIREY 15 175 EAINSDNYK 15 191PPTKQSLVK 15 220 LEHFGISEY 15 224 GISEYTMCL 15 236 YTMGLKNAR 15 250EAIDTESRL 15 281 TNSPLVPTF 15 302 SIALVSTNY 15 314 KTNSSSNDL 15 322LEVEDRTSL 15 326 DRTSLVLNS 15 347 PTISSYENL 15 381 NLATPIAIK 15 388IKAVPPSKR 15 397 FLKHGQNIR 15 11 LRSLASTLD 14 16 STLDCETAR 14 17TLDCETARL 14 52 QTLKDDVNI 14 61 PELSNCENF 14 83 VASSCISGK 14 114NPPQAVNLL 14 118 AVNLLDKAR 14 127 LENQEGIDF 14 129 NQEGIDFIK 14 145KNSMDIMKI 14 151 MKIREYFQK 14 159 KYGYSPRVK 14 186 PVIVTPPTK 14 197LVKVLKTPK 14 205 KCALKMDDF 14 212 DFECVTPKL 14 230 MCLNEDYTM 14 243ARNNKSEEA 14 255 ESRLNDNVF 14 256 SRLNDNVFA 14 272 QLEKSDAEY 14 297PSTKNSIAL 14 304 ALVSTNYPL 14 344 PSSPTISSY 14 349 ISSYENLLR 14 358TPTPPEVTK 14 387 AIKAVPPSK 14 390 AVPPSKRFL 14 404 IRDVSNKEN 14 2DPIRSFCGK 13 5 RSFCGKLRS 13 21 ETARLQRAL 13 39 YPMRILYDL 13 46 DLHSEVQTL13 63 LSNCENFQK 13 95 SPQLSDFGL 13 98 LSDFGLERY 13 149 DIMKIREYF 13 152KIREYFQKY 13 153 IREYFQKYG 13 157 FQKYGYSPR 13 180 DNYKEEPVI 13 189VTPPTKQSL 13 193 TKQSLVKVL 13 201 LKTPKCALK 13 214 ECVTPKLEH 13 215CVTPKLEHF 13 244 RNNKSEEAI 13 287 PTFCTPGLK 13 319 SNDLEVEDR 13 324VEDRTSLVL 13 356 LRTPTPPEV 13 359 PTPPEVTKI 13 367 IPEDILQLL 13 392PPSKRFLKH 13 3 PIRSFCGKL 12 19 DCETARLQR 12 26 QRALDGEES 12 37 EDYPMRILY12 70 QKTDVKDDL 12 97 QLSDFGLER 12 99 SDFGLERYI 12 116 PQAVNLLDK 12 128ENQEGIDFI 12 144 EKNSMDIMK 12 200 VLKTPKCAL 12 277 DAEYTNSPL 12 306VSTNYPLSK 12 333 NSDTCFENL 12 V5-B2705-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:11; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 2 VASSCISEK 15 5 SCISEKSPR 15 9 EKSPRSPQL 14V6-B2705-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:13; eachstart position is specified, the length of peptide is 9 amino acids, andthe end position for each peptide is the start position plus eight. 5EAIDAESRL 15 4 EEAIDAESR 12 V10-B2705-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:21; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart positon plus eight. 1 KIPEDILQK 18 2 IPEDILQKF 15 11 QWIYPTQKL 1513 IYPTQKLNK 15 14 YPTQKLNKM 15 6 ILQKFQWIY 14 15 PTQKLNKMR 14 10FQWIYPTQK 13 5 DILQKFQWI 11 8 QKFQWIYPT 9 V12-B2705-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:25; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 1 RALDGEESL 19 5 GEESLLSKY 162 ALDGEESLL 15 4 DGEESLLSK 15

TABLE XXXI Pos 123456789 score V1-HLA-B2709-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 104 ERYIVSQVL 22 125 ARLENQEGI21 356 LRTPTPPEV 21 93 PRSPQLSDF 19 90 GKSPRSPQL 16 23 ARLQRALDG 15 326DRTSLVLNS 15 366 KIPEDILQL 15 395 KRFLKHGQN 15 396 RFLKHGQNI 15 10KLRSLASTL 14 113 PNPPQAVNL 14 119 VNLLDKARL 14 256 SRLNDNVFA 14 304ALVSTNYPL 14 52 QTLKDDVNI 13 55 KDDVNIPEL 13 224 GISEYTMCL 13 265TPSPIIQQL 13 314 KTNSSSNDL 13 347 PTISSYENL 13 389 KAVPPSKRF 13 39YPMRILYDL 12 41 MRILYDLHS 12 46 DLHSEVQTL 12 61 PELSNCENF 12 133IDFIKATKV 12 140 KVLMEKNSM 12 158 QKYGYSPRV 12 193 TKQSLVKVL 12 202KTPKCALKM 12 244 RNNKSEEAI 12 250 EAIDTESRL 12 278 AEYTNSPLV 12 286VPTFCTPGL 12 322 LEVEDRTSL 12 367 IPEDILQLL 12 390 AVPPSKRFL 12 3PIRSFCGKL 11 4 IRSFCGKLR 11 17 TLDCETARL 11 26 QRALDGEES 11 43 ILYDLHSEV11 70 QKTDVKDDL 11 75 KDDLSDPPV 11 103 LERYIVSQV 11 114 NPPQAVNLL 11 134DFIKATKVL 11 145 KNSMDIMKI 11 153 IREYFQKYG 11 164 PRVKKNSVH 11 180DNYKEEPVI 11 189 VTPPTKQSL 11 212 DFECVTPKL 11 230 MCLNEDYTM 11 243ARNNKSEEA 11 281 TNSPLVPTF 11 295 KIPSTKNSI 11 297 PSTKNSIAL 11 324VEDRTSLVL 11 333 NSDTCFENL 11 348 TISSYENLL 11 374 LLSKYNSNL 11 404IRDVSNKEN 11 6 SFCGKLRSL 10 11 LRSLASTLD 10 21 ETARLQRAL 10 36 FEDYPMRIL10 95 SPQLSDFGL 10 99 SDFGLERYI 10 200 VLKTPKCAL 10 205 KCALKMDDF 10 215CVTPKLEHF 10 232 LNEDYTMGL 10 261 NVFATPSPI 10 277 DAEYTNSPL 10 316NSSSNDLEV 10 330 LVLNSDTCF 10 341 LTDPSSPTI 10 359 PTPPEVTKI 10 363EVTKIPEDI 10 364 VTKIPEDIL 10 380 SNLATPIAI 10 399 KHGQNIRDV 10V5-HLA-B2709-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:11;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 9 EKSPRSPQL 13 V6-HLA-B2709-9mers-193P1E1B Each peptide is aportion of SEQ ID NO:13; each start position is specified, the length ofpeptide is 9 amino acids, and the end position for each peptide is thestart position plus eight. 5 EAIDAESRL 12 9 AESRLNDNV 10V10-HLA-B2709-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 11 QWIYPTQKL 11 2 IPEDILQKF 10 5 DILQKFQWI 10 14 YPTQKLNKM 9 1KIPEDILQK 5 V12-HLA-B2709-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:25; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 1 RALDGEESL 16 2 ALDGEESLL 11

TABLE XXXII Pos 123456789 score V1-HLA-B4402-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 324 VEDRTSLVL 24 220 LEHFGISEY23 36 FEDYPMRIL 22 61 PELSNCENF 22 31 GEESDFEDY 21 127 LENQEGIDF 21 174QEAINSDNY 21 322 LEVEDRTSL 21 154 REYFQKYGY 20 183 KEEPVIVTP 19 265TPSPIIQQL 19 37 EDYPMRILY 18 390 AVPPSKRFL 18 134 DFIKATKVL 17 148MDIMKIREY 17 344 PSSPTISSY 17 366 KIPEDILQL 17 21 ETARLQRAL 16 55KDDVNIPEL 16 90 GKSPRSPQL 16 113 PNPPQAVNL 16 250 EAIDTESRL 16 255ESRLNDNVF 16 278 AEYTNSPLV 16 281 TNSPLVPTF 16 389 KAVPPSKRF 16 28ALDGEESDF 15 93 PRSPQLSDF 15 114 NPPQAVNLL 15 184 EEPVIVTPP 15 6SFCGKLRSL 14 32 EESDFEDYP 14 39 YPMRILYDL 14 49 SEVQTLKDD 14 145KNSMDIMKI 14 172 HEQEAINSD 14 189 VTPPTKQSL 14 193 TKQSLVKVL 14 213FECVTPKLE 14 215 CVTPKLEHF 14 297 PSTKNSIAL 14 333 NSDTCFENL 14 359PTPPEVTKI 14 362 PEVTKIPED 14 367 IPEDILQLL 14 380 SNLATPIAI 14 10KLRSLASTL 13 99 SDFGLERYI 13 103 LERYIVSQV 13 104 ERYIVSQVL 13 125ARLENQEGI 13 128 ENQEGIDFI 13 130 QEGIDFIKA 13 131 EGIDFIKAT 13 149DIMKIREYF 13 152 KIREYFQKY 13 200 VLKTPKCAL 13 226 SEYTMCLNE 13 233NEDYTMGLK 13 249 EEAIDTESR 13 304 ALVSTNYPL 13 341 LTDPSSPTI 13 347PTISSYENL 13 348 TISSYENLL 13 368 PEDILQLLS 13 370 DILQLLSKY 13 17TLDCETARL 12 20 CETARLQRA 12 46 DLHSEVQTL 12 95 SPQLSDFGL 12 98LSDFGLERY 12 119 VNLLDKARL 12 205 KCALKMDDF 12 212 DFECVTPKL 12 224GISEYTMCL 12 232 LNEDYTMGL 12 248 SEEAIDTES 12 254 TESRLNDNV 12 261NVFATPSPI 12 302 SIALVSTNY 12 314 KTNSSSNDL 12 330 LVLNSDTCF 12 352YENLLRTPT 12 363 EVTKIPEDI 12 3 PIRSFCGKL 11 70 QKTDVKDDL 11 143MEKNSMDIM 11 169 NSVHEQEAI 11 228 YTMCLNEDY 11 273 LEKSDAEYT 11 286VPTFCTPGL 11 288 TFCTPGLKI 11 295 KIPSTKNSI 11 338 FENLTDPSS 11 374LLSKYNSNL 11 378 YNSNLATPI 11 383 ATPIAIKAV 11V5-HLA-B4402-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:11;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 9 EKSPRSPQL 18 8 SEKSPRSPQ 12 V6-HLA-B4402-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:13; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 5 EAIDAESRL 16 9 AESRLNDNV 154 EEAIDAESR 13 3 SEEAIDAES 12 V10-HLA-B4402-9mers-193P1E1B Each peptideis a portion of SEQ ID NO:21; each start position is specified, thelength of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 4 EDILQKFQW 17 11 QWIYPTQKL 152 IPEDILQKF 14 3 PEDILQKFQ 13 5 DILQKFQWI 10 6 ILQKFQWIY 10V12-HLA-B4402-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 GEESLLSKY 22 2 ALDGEESLL 16 6 EESLLSKYN 16 1 RALDGEESL 12

TABLE XXXIII Pos 123456789 score V1-HLA-B5101-9mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 9 amino acids, and the end position for eachpeptide is the start position plus eight. 80 DPPVASSCI 25 180 DNYKEEPVI22 190 TPPTKQSLV 22 277 DAEYTNSPL 22 114 NPPQAVNLL 21 217 TPKLEHFGI 21163 SPRVKKNSV 20 367 IPEDILQLL 20 39 YPMRILYDL 19 250 EAIDTESRL 19 35DFEDYPMRI 18 265 TPSPIIQQL 18 95 SPQLSDFGL 17 286 VPTFCTPGL 17 360TPPEVTKIP 17 2 DPIRSFCGK 16 133 IDFIKATKV 16 303 IALVSTNYP 16 310YPLSKTNSS 16 359 PTPPEVTKI 16 380 SNLATPIAI 16 382 LATPIAIKA 16 43ILYDLHSEV 15 101 FGLERYIVS 15 104 ERYIVSQVL 15 112 LPNPPQAVN 15 115PPQAVNLLD 15 134 DFIKATKVL 15 158 QKYGYSPRV 15 160 YGYSPRVKK 15 191PPTKQSLVK 15 261 NVFATPSPI 15 288 TFCTPGLKI 15 296 IPSTKNSIA 15 341LTDPSSPTI 15 343 DPSSPTISS 15 384 TPIAIKAVP 15 386 IAIKAVPPS 15 392PPSKRFLKH 15 46 DLHSEVQTL 14 52 QTLKDDVNI 14 60 IPELSNCEN 14 125ARLENQEGI 14 137 KATKVLMEK 14 206 CALKMDDFE 14 212 DFECVTPKL 14 263FATPSPIIQ 14 358 TPTPPEVTK 14 378 YNSNLATPI 14 14 LASTLDCET 13 27RALDGEESD 13 30 DGEESDFED 13 99 SDFGLERYI 13 117 QAVNLLDKA 13 128ENQEGIDFI 13 142 LMEKNSMDI 13 145 KNSMDIMKI 13 181 NYKEEPVIV 13 192PTKQSLVKV 13 193 TKQSLVKVL 13 203 TPKCALKMD 13 278 AEYTNSPLV 13 291TPGLKIPST 13 295 KIPSTKNSI 13 391 VPPSKRFLK 13 396 RFLKHGQNI 13 81PPVASSCIS 12 83 VASSCISGK 12 100 DFGLERYIV 12 103 LERYIVSQV 12 175EAINSDNYK 12 185 EPVIVTPPT 12 208 LKMDDFECV 12 238 MGLKNARNN 12 244RNNKSEEAI 12 262 VFATPSPII 12 283 SPLVPTFCT 12 292 PGLKIPSTK 12 324VEDRTSLVL 12 356 LRTPTPPEV 12 361 PPEVIKIPE 12 363 EVTKIPEDI 12 389KAVPPSKRF 12 V5-HLA-B5101-9mers-193P1E1B Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is9 amino acids, and the end position for each peptide is the startposition plus eight. 2 VASSCISEK 12 7 ISEKSPRSP 6 9 EKSPRSPQL 6V6-HLA-B5101-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 EAIDAESRL 18 8 DAESRLNDN 16 9 AESRLNDNV 9V10-HLA-B5101-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 5 DILQKFQWI 18 14 YPTQKLNKM 17 2 IPEDILQKF 16 11 QWIYPTQKL 8V12-HLA-B5101-9mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 9 aminoacids, and the end position for each peptide is the start position pluseight. 1 RALDGEESL 19 4 DGEESLLSK 15

TABLE XXXIV Pos 1234567890 score V1-HLA-A1-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 36 FEDYPMRILY 33 30 DGEESDFEDY28 147 SMDIMKIREY 27 219 KLEHFGISEY 26 153 IREYFQKYGY 25 173 EQEAINSDNY25 71 KTDVKDDLSD 22 129 NQEGIDFIKA 20 151 MKIREYFQKY 20 225 ISEYTMCLNE20 341 LTDPSSPTIS 20 233 NEDYTMGLKN 19 301 NSIALVSTNY 19 78 LSDPPVASSC18 251 AIDTESRLND 18 367 IPEDILQLLS 18 253 DTESRLNDNV 17 323 EVEDRTSLVL17 369 EDILQLLSKY 17 97 QLSDFGLERY 16 126 RLENQEGIDF 16 227 EYTMCLNEDY16 333 NSDTCFENLT 16 368 PEDILQLLSK 16 V5-HLA-A1-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:11; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 8 ISEKSPRSPQ 15V6-HLA-A1-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 7 AIDAESRLND 18 3 KSEEAIDAES 14 4 SEEAIDAESR 12 9 DAESRLNDNV 11V10-HLA-A1-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 6 DILQKFQWIY 15 1 TKIPEDILQK 10 3 IPEDILQKFQ 10 4 PEDILQKFQW 10 13WIYPTQKLNK 10 V12-HLA-A1-10mers-193P1E1B Each peptide is a portion ofSEQ ID NO:25; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. 5 DGEESLLSKY 27 3 ALDGEESLLS 21

TABLE XXXV Pos 1234567890 score V1-HLA-A0201-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 355 LLRTPTPPEV 25 366KIPEDILQLL 25 102 GLERYIVSQV 24 231 CLNEDYTMGL 24 141 VLMEKNSMDI 22 373QLLSKYNSNL 22 16 STLDCETARL 21 42 RILYDLHSEV 21 207 ALKMDDFECV 21 340NLTDPSSPTI 21 382 LATPIAIKAV 21 13 SLASTLDCET 20 45 YDLHSEVQTL 20 54LKDDVNIPEL 20 132 GIDFIKATKV 20 264 ATPSPIIQQL 20 321 DLEVEDRTSL 20 110QVLPNPPQAV 19 118 AVNLLDKARL 19 188 IVTPPTKQSL 19 303 IALVSTNYPL 19 365TKIPEDILQL 19 77 DLSDPPVASS 18 199 KVLKTPKCAL 18 285 LVPTFCTPGL 18 381NLATPIAIKA 18 398 LKHGQNIRDV 18 5 RSFCGKLRSL 17 112 LPNPPQAVNL 17 189VTPPTKQSLV 17 290 CTPGLKIPST 17 294 LKIPSTKNSI 17 374 LLSKYNSNLA 17 389KAVPPSKRFL 17 99 SDFGLERYIV 16 120 NLLDKARLEN 16 121 LLDKARLENQ 16 147SMDIMKIREY 16 219 KLEHFGISEY 16 257 RLNDNVFATP 16 276 SDAEYTNSPL 16 9GKLRSLASTL 15 97 QLSDFGLERY 15 106 YIVSQVLPNP 15 127 LENQEGIDFI 15 162YSPRVKKNSV 15 211 DDFECVTPKL 15 229 TMCLNEDYTM 15 358 TPTPPEVTKI 15 28ALDGEESDFE 14 38 DYPMRILYDL 14 43 ILYDLHSEVQ 14 113 PNPPQAVNLL 14 124KARLENQEGI 14 191 PPTKQSLVKV 14 209 KMDDFECVTP 14 216 VTPKLEHFGI 14 261NVFATPSPII 14 269 IIQQLEKSDA 14 280 YTNSPLVPTF 14 322 LEVEDRTSLV 14 331VLNSDTCFEN 14 347 PTISSYENLL 14 2 DPIRSFCGKL 13 34 SDFEDYPMRI 13 74VKDDLSDPPV 13 101 FGLERYIVSQ 13 111 VLPNPPQAVN 13 135 FIKATKVLME 13 142LMEKNSMDIM 13 192 PTKQSLVKVL 13 223 FGISEYTMCL 13 239 GLKNARNNKS 13 256SRLNDNVFAT 13 272 QLEKSDAEYT 13 287 PTFCTPGLKI 13 295 KIPSTKNSIA 13 296IPSTKNSIAL 13 299 TKNSIALVST 13 302 SIALVSTNYP 13 304 ALVSTNYPLS 13 315TNSSSNDLEV 13 332 LNSDTCFENL 13 354 NLLRTPTPPE 13 371 ILQLLSKYNS 13 14LASTLDCETA 12 24 RLQRALDGEE 12 49 SEVQTLKDDV 12 59 NIPELSNCEN 12 79SDPPVASSCI 12 87 CISGKSPRSP 12 89 SGKSPRSPQL 12 133 IDFIKATKVL 12 144EKNSMDIMKI 12 176 AINSDNYKEE 12 180 DNYKEEPVIV 12 194 KQSLVKVLKT 12 196SLVKVLKTPK 12 200 VLKTPKCALK 12 243 ARNNKSEEAI 12 253 DTESRLNDNV 12 277DAEYTNSPLV 12 297 PSTKNSIALV 12 313 SKTNSSSNDL 12 323 EVEDRTSLVL 12 329SLVLNSDTCF 12 385 PIAIKAVPPS 12 V5-HLA-A0201-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:11; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 7 CISEKSPRSP 12 9 SEKSPRSPQL 122 PVASSCISEK 9 3 VASSCISEKS 7 6 SCISEKSPRS 5V6-HLA-A0201-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 9 DAESRLNDNV 12 7 AIDAESRLND 11 8 IDAESRLNDN 11 5 EEAIDAESRL 9 2NKSEEAIDAE 7 3 KSEEAIDAES 6 V10-HLA-A0201-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:21; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 2 KIPEDILQKF 17 11 FQWIYPTQKL14 14 IYPTQKLNKM 13 7 ILQKFQWIYP 12 13 WIYPTQKLNK 12V12-HLA-A0201-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 9 SLLSKYNSNL 24 2 RALDGEESLL 17 3 ALDGEESLLS 15 1 QRALDGEESL 14 4LDGEESLLSK 11

TABLE XXXVI Pos 1234567890 score V1-HLA-A0203-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 6 SFCGKLRSLA 10 14 LASTLDCETA10 19 DCETARLQRA 10 75 KDDLSDPPVA 10 109 SQVLPNPPQA 10 116 PQAVNLLDKA 10129 NQEGIDFIKA 10 167 KKNSVHEQEA 10 198 VKVLKTPKCA 10 234 EDYTMGLKNA 10242 NARNNKSEEA 10 255 ESRLNDNVFA 10 269 IIQQLEKSDA 10 295 KIPSTKNSIA 10374 LLSKYNSNLA 10 378 YNSNLATPIA 10 381 NLATPIAIKA 10 7 FCGKLRSLAS 9 15ASTLDCETAR 9 20 CETARLQRAL 9 76 DDLSDPPVAS 9 110 QVLPNPPQAV 9 117QAVNLLDKAR 9 130 QEGIDFIKAT 9 168 KNSVHEQEAI 9 199 KVLKTPKCAL 9 235DYTMGLKNAR 9 243 ARNNKSEEAI 9 256 SRLNDNVFAT 9 270 IQQLEKSDAE 9 296IPSTKNSIAL 9 375 LSKYNSNLAT 9 379 NSNLATPIAI 9 382 LATPIAIKAV 9 8CGKLRSLAST 8 16 STLDCETARL 8 21 ETARLQRALD 8 77 DLSDPPVASS 8 111VLPNPPQAVN 8 118 AVNLLDKARL 8 131 EGIDFIKATK 8 169 NSVHEQEAIN 8 200VLKTPKCALK 8 236 YTMGLKNARN 8 244 RNNKSEEAID 8 257 RLNDNVFATP 8 271QQLEKSDAEY 8 297 PSTKNSIALV 8 376 SKYNSNLATP 8 380 SNLATPIAIK 8 383ATPIAIKAVP 8 V5-HLA-A0203-10mers-193P1E1B NoResultsFound.V6-HLA-A0203-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 1 NNKSEEAIDA 10 2 NKSEEAIDAE 9 3 KSEEAIDAES 8V10-HLA-A0203-10mers-193P1E1B NoResultsFound.V12-HLA-A0203-10mers-193P1E1B NoResultsFound.

TABLE XXXVII Pos 1234567890 score V1-HLA-A3-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:3; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 390 AVPPSKRFLK 27 305LVSTNYPLSK 26 62 ELSNCENFQK 24 82 PVASSCISGK 24 200 VLKTPKCALK 24 158QKYGYSPRVK 23 126 RLENQEGIDF 22 196 SLVKVLKTPK 22 219 KLEHFGISEY 22 257RLNDNVFATP 22 357 RTPTPPEVTK 22 387 AIKAVPPSKR 22 43 ILYDLHSEVQ 21 46DLHSEVQTLK 21 131 EGIDFIKATK 21 97 QLSDFGLERY 19 102 GLERYIVSQV 19 110QVLPNPPQAV 19 140 KVLMEKNSMD 19 291 TPGLKIPSTK 19 3 PIRSFCGKLR 18 24RLQRALDGEE 18 111 VLPNPPQAVN 18 159 KYGYSPRVKK 18 323 EVEDRTSLVL 18 380SNLATPIAIK 18 386 IAIKAVPPSK 18 10 KLRSLASTLD 17 42 RILYDLHSEV 17 118AVNLLDKARL 17 120 NLLDKARLEN 17 150 IMKIREYFQK 17 188 IVTPPTKQSL 17 190TPPTKQSLVK 17 329 SLVLNSDTCF 17 373 QLLSKYNSNL 17 77 DLSDPPVASS 16 185EPVIVTPPTK 16 199 KVLKTPKCAL 16 368 PEDILQLLSK 16 9 GKLRSLASTL 15 28ALDGEESDFE 15 115 PPQAVNLLDK 15 135 FIKATKVLME 15 152 KIREYFQKYG 15 163SPRVKKNSVH 15 165 RVKKNSVHEQ 15 170 SVHEQEAINS 15 186 PVIVTPPTKQ 15 251AIDTESRLND 15 272 QLEKSDAEYT 15 278 AEYTNSPLVP 15 311 PLSKTNSSSN 15 340NLTDPSSPTI 15 348 TISSYENLLR 15 354 NLLRTPTPPE 15 27 RALDGEESDF 14 50EVQTLKDDVN 14 57 DVNIPELSNC 14 66 CENFQKTDVK 14 136 IKATKVLMEK 14 174QEAINSDNYK 14 193 TKQSLVKVLK 14 207 ALKMDDFECV 14 266 PSPIIQQLEK 14 271QQLEKSDAEY 14 284 PLVPTFCTPG 14 295 KIPSTKNSIA 14 321 DLEVEDRTSL 14 355LLRTPTPPEV 14 381 NLATPIAIKA 14 401 GQNIRDVSNK 14 17 TLDCETARLQ 13 87CISGKSPRSP 13 107 IVSQVLPNPP 13 143 MEKNSMDIMK 13 215 CVTPKLEHFG 13 238MGLKNARNNK 13 268 PIIQQLEKSD 13 269 IIQQLEKSDA 13 298 STKNSIALVS 13 304ALVSTNYPLS 13 330 LVLNSDTCFE 13 369 EDILQLLSKY 13 371 ILQLLSKYNS 13 376SKYNSNLATP 13 V5-HLA-A3-10mers-193P1E1B Each peptide is a portion of SEQID NO:11; each start position is specified, the length of peptide is 10amino acids, and the end position for each peptide is the start positionplus nine. 2 PVASSCISEK 24 7 CISEKSPRSP 12 V6-HLA-A3-10mers-193P1E1BEach peptide is a portion of SEQ ID NO:13; each start position isspecified, the length of peptide is 10 amino acids, and the end positionfor each peptide is the start position plus nine. 7 AIDAESRLND 17 10AESRLNDNVF 14 4 SEEAIDAESR 12 3 KSEEAIDAES V10-HLA-A3-10mers-193P1E1BEach peptide is a portion of SEQ ID NO:21; each start position isspecified, the length of peptide is 10 amino acids, and the end positionfor each peptide is the start position plus nine. 13 WIYPTQKLNK 28 1TKIPEDILQK 22 10 KFQWIYPTQK 18 2 KIPEDILQKF 16 6 DILQKFQWIY 16V12-HLA-A3-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 3 ALDGEESLLS 18 4 LDGEESLLSK 16 9 SLLSKYNSNL 16 2 RALDGEESLL 10 5DGEESLLSKY 10

TABLE XXXVIII Pos 1234567890 score V1-HLA-A26-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 323 EVEDRTSLVL 30 369EDILQLLSKY 30 363 EVTKIPEDIL 29 214 ECVTPKLEHF 26 343 DPSSPTISSY 25 57DVNIPELSNC 23 211 DDFECVTPKL 23 2 DPIRSFCGKL 22 264 ATPSPIIQQL 22 280YTNSPLVPTF 22 188 IVTPPTKQSL 21 227 EYTMCLNEDY 21 347 PTISSYENLL 21 30DGEESDFEDY 20 38 DYPMRILYDL 20 50 EVQTLKDDVN 20 173 EQEAINSDNY 20 192PTKQSLVKVL 20 335 DTCFENLTDP 20 365 TKIPEDILQL 20 73 DVKDDLSDPP 19 199KVLKTPKCAL 19 249 EEAIDTESRL 19 16 STLDCETARL 18 21 ETARLQRALD 18 118AVNLLDKARL 18 285 LVPTFCTPGL 18 366 KIPEDILQLL 18 5 RSFCGKLRSL 17 35DFEDYPMRIL 17 253 DTESRLNDNV 17 321 DLEVEDRTSL 17 37 EDYPMRILYD 16 82PVASSCISGK 16 131 EGIDFIKATK 16 144 EKNSMDIMKI 16 155 EYFQKYGYSP 16 67ENFQKTDVKD 15 92 SPRSPQLSDF 15 97 QLSDFGLERY 15 147 SMDIMKIREY 15 151MKIREYFQKY 15 175 EAINSDNYKE 15 185 EPVIVTPPTK 15 186 PVIVTPPTKQ 15 219KLEHFGISEY 15 221 EHFGISEYTM 15 250 EAIDTESRLN 15 279 EYTNSPLVPT 15 287PTFCTPGLKI 15 325 EDRTSLVLNS 15 326 DRTSLVLNSD 15 56 DDVNIPELSN 14 77DLSDPPVASS 14 165 RVKKNSVHEQ 14 170 SVHEQEAINS 14 234 EDYTMGLKNA 14 261NVFATPSPII 14 301 NSIALVSTNY 14 V5-HLA-A26-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:11; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 2 PVASSCISEK 16 9 SEKSPRSPQL 1110 EKSPRSPQLS 11 7 CISEKSPRSP 7 V6-HLA-A26-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:13; each start position is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 5 EEAIDAESRL 19 6 EAIDAESRLN 159 DAESRLNDNV 9 V10-HLA-A26-10mers-193P1E1B Each peptide is a portion ofSEQ ID NO:21; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. 6 DILQKFQWIY 22 2 KIPEDILQKF 19 5 EDILQKFQWI 14 1TKIPEDILQK 12 V12-HLA-A26-10mers-193P1E1B Each peptide is a portion ofSEQ ID NO:25; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. 5 DGEESLLSKY 26

TABLE XXXIX Pos 1234567890 score V1-HLA-B0702-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 296 IPSTKNSIAL 24 112LPNPPQAVNL 23 2 DPIRSFCGKL 20 346 SPTISSYENL 20 191 PPTKQSLVKV 19 358TPTPPEVTKI 19 92 SPRSPQLSDF 18 60 IPELSNCENF 17 323 EVEDRTSLVL 14 384TPIAIKAVPP 14 103 LERYIVSQVL 13 115 PPQAVNLLDK 13 118 AVNLLDKARL 13 133IDFIKATKVL 13 163 SPRVKKNSVH 13 190 TPPTKQSLVK 13 199 KVLKTPKCAL 13 265TPSPIIQQLE 13 332 LNSDTCFENL 13 365 TKIPEDILQL 13 367 IPEDILQLLS 13 389KAVPPSKRFL 13 391 VPPSKRFLKH 13 392 PPSKRFLKHG 13 16 STLDCETARL 12 20CETARLQRAL 12 54 LKDDVNIPEL 12 113 PNPPQAVNLL 12 114 NPPQAVNLLD 12 185EPVIVTPPTK 12 188 IVTPPTKQSL 12 192 PTKQSLVKVL 12 194 KQSLVKVLKT 12 211DDFECVTPKL 12 249 EEAIDTESRL 12 255 ESRLNDNVFA 12 264 ATPSPIIQQL 12 276SDAEYTNSPL 12 285 LVPTFCTPGL 12 303 IALVSTNYPL 12 321 DLEVEDRTSL 12 343DPSSPTISSY 12 363 EVTKIPEDIL 12 5 RSFCGKLRSL 11 39 YPMRILYDLH 11 45YDLHSEVQTL 11 81 PPVASSCISG 11 89 SGKSPRSPQL 11 94 RSPQLSDFGL 11 95SPQLSDFGLE 11 217 TPKLEHFGIS 11 223 FGISEYTMCL 11 231 CLNEDYTMGL 11 283SPLVPTFCTP 11 291 TPGLKIPSTK 11 349 ISSYENLLRT 11 360 TPPEVTKIPE 11 361PPEVTKIPED 11 366 KIPEDILQLL 11 V5-HLA-B0702-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:11; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 1 PPVASSCISE 11 9 SEKSPRSPQL 11V6-HLA-B0702-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 5 EEAIDAESRL 12 10 AESRLNDNVF 11 7 AIDAESRLND 7 1 NNKSEEAIDA 6 9DAESRLNDNV 6 V10-HLA-B0702-10mers-193P1E1B Each peptide is a portion ofSEQ ID NO:21; each start position is specified, the length of peptide is10 amino acids, and the end position for each peptide is the startposition plus nine. 3 IPEDILQKFQ 12 11 FQWIYPTQKL 11 15 YPTQKLNKMR 10 5EDILQKFQWI 8 2 KIPEDILQKF 7 8 LQKFQWIYPT 7 14 IYPTQKLNKM 7V12-HLA-B0702-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:25;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 1 QRALDGEESL 11 2 RALDGEESLL 11 9 SLLSKYNSNL 10 3 ALDGEESLLS 7

TABLE XL Pos 1234567890 score V1-HLA-B08-10mers-193P1E1B NoResultsFound.V5-HLA-B08-10mers-193P1E1B NoResultsFound. V6-HLA-B08-10mers-193P1E1BNoResultsFound. V10-HLA-B08-10mers-193P1E1B NoResultsFound.V12-HLA-B08-10mers-193P1E1B NoResultsFound.

TABLE XLI Pos 1234567890 score V1-HLA-B1510-10mers-193P1E1BNoResultsFound. V5-HLA-B1510-10mers-193P1E1B NoResultsFound.V6-HLA-B1510-10mers-193P1E1B NoResultsFound.V10-HLA-B1510-10mers-193P1E1B NoResultsFound.V12-HLA-B1510-10mers-193P1E1B NoResultsFound.

TABLE XLII Pos 1234567890 score V1-HLA-B2705-10mers-193P1E1BNoResultsFound. V5-HLA-B2705-10mers-193P1E1B NoResultsFound.V6-HLA-B2705-10mers-193P1E1B NoResultsFound.V10-HLA-B2705-10mers-193P1E1B NoResultsFound.V12-HLA-B2705-10mers-193P1E1B NoResultsFound.

TABLE XLIII Pos 1234567890 score V1-HLA-B2709-10mers-193P1E1BNoResultsFound. V5-HLA-B2709-10mers-193P1E1B NoResultsFound.V6-HLA-B2709-10mers-193P1E1B NoResultsFound.V10-HLA-B2709-10mers-193P1E1B NoResultsFound.V12-HLA-B2709-10mers-193P1E1B NoResultsFound.

TABLE XLIV Pos 1234567890 score V1-HLA-B4402-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:3; each start position is specified,the length of peptide is 10 amino acids, and the end postion for eachpeptide is the start position plus nine. 36 FEDYPMRILY 24 254 TESRLNDNVF24 20 CETARLQRAL 23 249 EEAIDTESRL 22 103 LERYIVSQVL 21 127 LENQEGIDFI21 365 TKIPEDILQL 21 362 PEVTKIPEDI 20 264 ATPSPIIQQL 18 369 EDILQLLSKY18 113 PNPPQAVNLL 17 130 QEGIDFIKAT 17 278 AEYTNSPLVP 17 133 IDFIKATKVL16 147 SMDIMKIREY 16 151 MKIREYFQKY 16 2 DPIRSFCGKL 15 54 LKDDVNIPEL 15112 LPNPPQAVNL 15 183 KEEPVIVTPP 15 294 LKIPSTKNSI 15 296 IPSTKNSIAL 15323 EVEDRTSLVL 15 324 VEDRTSLVLN 15 347 PTISSYENLL 15 389 KAVPPSKRFL 155 RSFCGKLRSL 14 9 GKLRSLASTL 14 16 STLDCETARL 14 32 EESDFEDYPM 14 118AVNLLDKARL 14 144 EKNSMDIMKI 14 148 MDIMKIREYF 14 184 EEPVIVTPPT 14 192PTKQSLVKVL 14 199 KVLKTPKCAL 14 211 DDFECVTPKL 14 214 ECVTPKLEHF 14 219KLEHFGISEY 14 223 FGISEYTMCL 14 226 SEYTMCLNED 14 233 NEDYTMGLKN 14 243ARNNKSEEAI 14 301 NSIALVSTNY 14 343 DPSSPTISSY 14 366 KIPEDILQLL 14 379NSNLATPIAI 14 45 YDLHSEVQTL 13 79 SDPPVASSCI 13 97 QLSDFGLERY 13 188IVTPPTKQSL 13 285 LVPTFCTPGL 13 313 SKTNSSSNDL 13 332 LNSDTCFENL 13 352YENLLRTPTP 13 358 TPTPPEVTKI 13 368 PEDILQLLSK 13 27 RALDGEESDF 12 34SDFEDYPMRI 12 35 DFEDYPMRIL 12 38 DYPMRILYDL 12 61 PELSNCENFQ 12 66CENFQKTDVK 12 89 SGKSPRSPQL 12 92 SPRSPQLSDF 12 126 RLENQEGIDF 12 143MEKNSMDIMK 12 168 KNSVHEQEAI 12 173 EQEAINSDNY 12 227 EYTMCLNEDY 12 248SEEAIDTESR 12 280 YTNSPLVPTF 12 287 PTFCTPGLKI 12 322 LEVEDRTSLV 12 329SLVLNSDTCF 12 338 FENLTDPSSP 12 363 EVTKIPEDIL 12 388 IKAVPPSKRF 12 395KRFLKHGQNI 12 30 DGEESDFEDY 11 49 SEVQTLKDDV 11 60 IPELSNCENF 11 69FQKTDVKDDL 11 94 RSPQLSDFGL 11 174 QEAINSDNYK 11 213 FECVTPKLEH 11 231CLNEDYTMGL 11 261 NVFATPSPII 11 271 QQLEKSDAEY 11 273 LEKSDAEYTN 11 276SDAEYTNSPL 11 303 IALVSTNYPL 11 321 DLEVEDRTSL 11 340 NLTDPSSPTI 11 346SPTISSYENL 11 373 QLLSKYNSNL 11 V5-HLA-B4402-10mers-193P1E1B Eachpeptide is a portion of SEQ ID NO:11; each start position is specified,the length of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 9 SEKSPRSPQL 22V6-HLA-B4402-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:13;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start postion plusnine. 10 AESRLNDNVF 27 5 EEAIDAESRL 22 4 SEEAIDAESR 12V10-HLA-B4402-10mers-193P1E1B Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 10 aminoacids, and the end position for each peptide is the start position plusnine. 4 PEDILQKFQW 22 5 EDILQKFQWI 15 2 KIPEDILQKF 14 11 FQWIYPTQKL 12 1TKIPEDILQK 11 6 DILQKFQWIY 11 V12-HLA-B4402-10mers-193P1E1B Each peptideis a portion of SEQ ID NO:25; each start postition is specified, thelength of peptide is 10 amino acids, and the end position for eachpeptide is the start position plus nine. 7 EESLLSKYNS 14 2 RALDGEESLL 135 DGEESLLSKY 12 6 GEESLLSKYN 12 9 SLLSKYNSNL 12 1 QRALDGEESL 11 3ALDGEESLLS 7

TABLE XLV Pos 123456789012345 score V1-HLA-B5101-10mers-193P1E1BNoResultsFound. V5-HLA-B5101-10mers-193P1E1B NoResultsFound.V6-HLA-B5101-10mers-193P1E1B NoResultsFound.V10-HLA-B5101-10mers-193P1E1B NoResultsFound.V12-HLA-B5101-10mers-193P1E1B NoResultsFound.

TABLE XLVI Pos 123456789012345 score V1-HLA-DRB1-0101-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:3; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 130QEGIDFIKATKVLME 33 124 KARLENQEGIDFIKA 31 168 KNSVHEQEAINSDNY 31 38DYPMRILYDLHSEVQ 29 300 KNSIALVSTNYPLSK 29 105 RYIVSQVLPNPPQAV 28 375LSKYNSNLATPIAIK 28 291 TPGLKIPSTKNSIAL 27 23 ARLQRALDGEESDFE 26 116PQAVNLLDKARLENQ 26 153 IREYFQKYGYSPRVK 26 335 DTCFENLTDPSSPTI 26 8CGKLRSLASTLDCET 25 229 TMCLNEDYTMGLKNA 25 267 SPIIQQLEKSDAEYT 25 307STNYPLSKTNSSSND 25 361 PPEVTKIPEDILQLL 25 369 EDILQLLSKYNSNLA 25 379NSNLATPIAIKAVPP 25 5 RSFCGKLRSLASTLD 24 185 EPVIVTPPTKQSLVK 24 195QSLVKVLKTPKCALK 24 283 SPLVPTFCTPGLKIP 24 350 SSYENLLRTPTPPEV 24 353ENLLRTPTPPEVTKI 24 372 LQLLSKYNSNLATPI 24 71 KTDVKDDLSDPPVAS 23 82PVASSCISGKSPRSP 23 85 SSCISGKSPRSPQLS 23 104 ERYIVSQVLPNPPQA 23 210MDDFECVTPKLEHFG 23 256 SRLNDNVFATPSPII 23 338 FENLTDPSSPTISSY 23 368PEDILQLLSKYNSNL 23 41 MRILYDLHSEVQTLK 22 101 FGLERYIVSQVLPNP 22 108VSQVLPNPPQAVNLL 22 139 TKVLMEKNSMDIMKI 22 259 NDNVFATPSPIIQQL 22 321DLEVEDRTSLVLNSD 22 376 SKYNSNLATPIAIKA 22 382 LATPIAIKAVPPSKR 22 385PIAIKAVPPSKRFLK 22 214 ECVTPKLEHFGISEY 21 77 DLSDPPVASSCISGK 20 132GIDFIKATKVLMEKN 20 286 VPTFCTPGLKIPSTK 20 1 MDPIRSFCGKLRSLA 19 33ESDFEDYPMRILYDL 19 36 FEDYPMRILYDLHSE 19 98 LSDFGLERYIVSQVL 19 159KYGYSPRVKKNSVHE 19 327 RTSLVLNSDTCFENL 19 349 ISSYENLLRTPTPPE 19 383ATPIAIKAVPPSKRF 19 4 IRSFCGKLRSLASTL 18 92 SPRSPQLSDFGLERY 18 131EGIDFIKATKVLMEK 18 136 IKATKVLMEKNSMDI 18 140 KVLMEKNSMDIMKIR 18 145KNSMDIMKIREYFQK 18 189 VTPPTKQSLVKVLKT 18 194 KQSLVKVLKTPKCAL 18 197LVKVLKTPKCALKMD 18 199 KVLKTPKCALKMDDF 18 205 KCALKMDDFECVTPK 18 227EYTMCLNEDYTMGLK 18 235 DYTMGLKNARNNKSE 18 237 TMGLKNARNNKSEEA 18 265TPSPIIQQLEKSDAE 18 270 IQQLEKSDAEYTNSP 18 15 ASTLDCETARLQRAL 17 20CETARLQRALDGEES 17 97 QLSDFGLERYIVSQV 17 102 GLERYIVSQVLPNPP 17 110QVLPNPPQAVNLLDK 17 137 KATKVLMEKNSMDIM 17 178 NSDNYKEEPVIVTPP 17 186PVIVTPPTKQSLVKV 17 196 SLVKVLKTPKCALKM 17 207 ALKMDDFECVTPKLE 17 255ESRLNDNVFATPSPI 17 285 LVPTFCTPGLKIPST 17 296 IPSTKNSIALVSTNY 17 302SIALVSTNYPLSKTN 17 306 VSTNYPLSKTNSSSN 17 319 SNDLEVEDRTSLVLN 17 393PSKRFLKHGQNIRDV 17 7 FCGKLRSLASTLDCE 16 12 RSLASTLDCETARLQ 16 40PMRILYDLHSEVQTL 16 48 HSEVQTLKDDVNIPE 16 57 DVNIPELSNCENFQK 16 72TDVKDDLSDPPVASS 16 75 KDDLSDPPVASSCIS 16 100 DFGLERYIVSQVLPN 16 107IVSQVLPNPPQAVNL 16 142 LMEKNSMDIMKIREY 16 147 SMDIMKIREYFQKYG 16 183KEEPVIVTPPTKQSL 16 184 EEPVIVTPPTKQSLV 16 202 KTPKCALKMDDFECV 16 232LNEDYTMGLKNARNN 16 240 LKNARNNKSEEAIDT 16 252 IDTESRLNDNVFATP 16 258LNDNVFATPSPIIQQ 16 260 DNVFATPSPIIQQLE 16 290 CTPGLKIPSTKNSIA 16 293GLKIPSTKNSIALVS 16 309 NYPLSKTNSSSNDLE 16 318 SSNDLEVEDRTSLVL 16 336TCFENLTDPSSPTIS 16 351 SYENLLRTPTPPEVT 16 364 VTKIPEDILQLLSKY 16 371ILQLLSKYNSNLATP 16 377 KYNSNLATPIAIKAV 16 380 SNLATPIAIKAVPPS 16V5-HLA-DRB1-0101-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:11; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 10 SSCISEKSPRSPQLS 23 2 DLSDPPVASSCISEK 20 6PPVASSCISEKSPRS 15 7 PVASSCISEKSPRSP 15 12 CISEKSPRSPQLSDF 15 3LSDPPVASSCISEKS 14 9 ASSCISEKSPRSPQL 14 13 ISEKSPRSPQLSDFG 14V6-HLA-DRB1-0101-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:13; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 4 ARNNKSEEAIDAESR 18 1 LKNARNNKSEEAIDA 16 13IDAESRLNDNVFATP 16 7 NKSEEAIDAESRLND 14 8 KSEEAIDAESRLNDN 11 14DAESRLNDNVFATPS 11 2 KNARNNKSEEAIDAE 9 6 NNKSEEAIDAESRLN 8 9SEEAIDAESRLNDNV 8 10 EEAIDAESRLNDNVF 8 12 AIDAESRLNDNVFAT 8V10-HLA-DRB1-0101-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:21; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 2 PPEVTKIPEDILQKF 25 13 LQKFQWIYPTQKLNK 23 10EDILQKFQWIYPTQK 18 14 QKFQWIYPTQKLNKM 18 5 VTKIPEDILQKFQWI 17 1TPPEVTKIPEDILQK 15 8 IPEDILQKFQWIYPT 15V12-HLA-DRB1-0101-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:25; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 3 ARLQRALDGEESLLS 26 13 ESLLSKYNSNLATPI 24 6QRALDGEESLLSKYN 23 10 DGEESLLSKYNSNLA 17 12 EESLLSKYNSNLATP 16 9LDGEESLLSKYNSNL 15

TABLE XLVII Pos 123456789012345 score V1-HLA-DRB1-0301-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:3; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 364VTKIPEDILQLLSKY 31 229 TMCLNEDYTMGLKNA 29 197 LVKVLKTPKCALKMD 28 371ILQLLSKYNSNLATP 28 51 VQTLKDDVNIPELSN 27 186 PVIVTPPTKQSLVKV 27 116PQAVNLLDKARLENQ 26 361 PPEVTKIPEDILQLL 26 67 ENFQKTDVKDDLSDP 25 247KSEEAIDTESRLNDN 24 319 SNDLEVEDRTSLVLN 24 40 PMRILYDLHSEVQTL 23 71KTDVKDDLSDPPVAS 22 205 KCALKMDDFECVTPK 22 174 QEAINSDNYKEEPVI 21 327RTSLVLNSDTCFENL 21 329 SLVLNSDTCFENLTD 21 138 ATKVLMEKNSMDIMK 20 346SPTISSYENLLRTPT 20 13 SLASTLDCETARLQR 19 95 SPQLSDFGLERYIVS 19 118AVNLLDKARLENQEG 19 124 KARLENQEGIDFIKA 19 145 KNSMDIMKIREYFQK 19 213FECVTPKLEHFGISE 19 217 TPKLEHFGISEYTMC 19 237 TMGLKNARNNKSEEA 19 249EEAIDTESRLNDNVF 19 321 DLEVEDRTSLVLNSD 19 369 EDILQLLSKYNSNLA 19 388IKAVPPSKRFLKHGQ 19 24 RLQRALDGEESDFED 18 44 LYDLHSEVQTLKDDV 18 57DVNIPELSNCENFQK 18 109 SQVLPNPPQAVNLLD 18 150 IMKIREYFQKYGYSP 18 194KQSLVKVLKTPKCAL 18 266 PSPIIQQLEKSDAEY 18 271 QQLEKSDAEYTNSPL 18 283SPLVPTFCTPGLKIP 18 293 GLKIPSTKNSIALVS 18 315 TNSSSNDLEVEDRTS 18 345SSPTISSYENLLRTP 18 394 SKRFLKHGQNIRDVS 18 18 LDCETARLQRALDGE 17 25LQRALDGEESDFEDY 17 29 LDGEESDFEDYPMRI 17 33 ESDFEDYPMRILYDL 17 60IPELSNCENFQKTDV 17 132 GIDFIKATKVLMEKN 17 225 ISEYTMCLNEDYTMG 17 267SPIIQQLEKSDAEYT 17 387 AIKAVPPSKRFLKHG 17 395 KRFLKHGQNIRDVSN 17 4IRSFCGKLRSLASTL 16 74 VKDDLSDPPVASSCI 16 108 VSQVLPNPPQAVNLL 16 146NSMDIMKIREYFQKY 16 147 SMDIMKIREYFQKYG 16 206 CALKMDDFECVTPKL 16 301NSIALVSTNYPLSKT 16 34 SDFEDYPMRILYDLH 15 117 QAVNLLDKARLENQE 15 274EKSDAEYTNSPLVPT 15 V5-HLA-DRB1-0301-15mers Each peptide is a portion ofSEQ ID NO:11; each start position is specified, the length of peptide is15 amino acids, and the end position for each peptide is the startposition plus fourteen. 10 SSCISEKSPRSPQLS 12 5 DPPVASSCISEKSPR 11 12CISEKSPRSPQLSDF 10 15 EKSPRSPQLSDFGLE 10 14 SEKSPRSPQLSDFGL 8 7PVASSCISEKSPRSP 7 8 VASSCISEKSPRSPQ 7 11 SCISEKSPRSPQLSD 7V6-HLA-DRB1-0301-15mers Each peptide is a portion of SEQ ID NO:13; eachstart position is specified, the length of peptide is 15 amino acids,and the end position for each peptide is the start position plusfourteen. 8 KSEEAIDAESRLNDN 24 10 EEAIDAESRLNDNVF 19 15 AESRLNDNVFATPSP13 V10-HLA-DRB1-0301-15mers Each peptide is a portion of SEQ ID NO:21;each start position is specified, the length of peptide is 15 aminoacids, and the end position for each peptide is the start position plusfourteen. 5 VTKIPEDILQKFQWI 31 2 PPEVTKIPEDILQKF 26 9 PEDILQKFQWIYPTQ 26V12-HLA-DRB1-0301-15mers Each peptide is a portion of SEQ ID NO:25; eachstart position is specified, the length of peptide is 15 amino acids,and the end position for each peptide is the start position plusfourteen. 12 EESLLSKYNSNLATP 28 4 RLQRALDGEESLLSK 26 5 LQRALDGEESLLSKY18 6 QRALDGEESLLSKYN 13

TABLE XLVIII Pos 123456789012345 score V1-HLA-DR1-0401-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:3; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 349ISSYENLLRTPTPPE 28 40 PMRILYDLHSEVQTL 26 41 MRILYDLHSEVQTLK 26 44LYDLHSEVQTLKDDV 26 57 DVNIPELSNCENFQK 26 229 TMCLNEDYTMGLKNA 26 237TMGLKNARNNKSEEA 26 283 SPLVPTFCTPGLKIP 26 319 SNDLEVEDRTSLVLN 26 368PEDILQLLSKYNSNL 26 98 LSDFGLERYIVSQVL 22 132 GIDFIKATKVLMEKN 22 157FQKYGYSPRVKKNSV 22 179 SDNYKEEPVIVTPPT 22 307 STNYPLSKTNSSSND 22 335DTCFENLTDPSSPTI 22 8 CGKLRSLASTLDCET 20 15 ASTLDCETARLQRAL 20 26QRALDGEESDFEDYP 20 38 DYPMRILYDLHSEVQ 20 48 HSEVQTLKDDVNIPE 20 51VQTLKDDVNIPELSN 20 60 IPELSNCENFQKTDV 20 71 KTDVKDDLSDPPVAS 20 109SQVLPNPPQAVNLLD 20 116 PQAVNLLDKARLENQ 20 119 VNLLDKARLENQEGI 20 130QEGIDFIKATKVLME 20 138 ATKVLMEKNSMDIMK 20 147 SMDIMKIREYFQKYG 20 185EPVIVTPPTKQSLVK 20 194 KQSLVKVLKTPKCAL 20 195 QSLVKVLKTPKCALK 20 205KCALKMDDFECVTPK 20 249 EEAIDTESRLNDNVF 20 259 NDNVFATPSPIIQQL 20 267SPIIQQLEKSDAEYT 20 291 TPGLKIPSTKNSIAL 20 293 GLKIPSTKNSIALVS 20 300KNSIALVSTNYPLSK 20 309 NYPLSKTNSSSNDLE 20 329 SLVLNSDTCFENLTD 20 338FENLTDPSSPTISSY 20 361 PPEVTKIPEDILQLL 20 364 VTKIPEDILQLLSKY 20 369EDILQLLSKYNSNLA 20 372 LQLLSKYNSNLATPI 20 388 IKAVPPSKRFLKHGQ 20 5RSFCGKLRSLASTLD 18 77 DLSDPPVASSCISGK 18 78 LSDPPVASSCISGKS 18 97QLSDFGLERYIVSQV 18 101 FGLERYIVSQVLPNP 18 106 YIVSQVLPNPPQAVN 18 122LDKARLENQEGIDFI 18 170 SVHEQEAINSDNYKE 18 182 YKEEPVIVTPPTKQS 18 214ECVTPKLEHFGISEY 18 221 EHFGISEYTMCLNED 18 234 EDYTMGLKNARNNKS 18 264ATPSPIIQQLEKSDA 18 280 YTNSPLVPTFCTPGL 18 290 CTPGLKIPSTKNSIA 18 320NDLEVEDRTSLVLNS 18 325 EDRTSLVLNSDTCFE 18 337 CFENLTDPSSPTISS 18 343DPSSPTISSYENLLR 18 365 TKIPEDILQLLSKYN 18 376 SKYNSNLATPIAIKA 18 392PPSKRFLKHGQNIRD 18 4 IRSFCGKLRSLASTL 17 33 ESDFEDYPMRILYDL 16 42RILYDLHSEVQTLKD 16 103 LERYIVSQVLPNPPQ 16 210 MDDFECVTPKLEHFG 16 225ISEYTMCLNEDYTMG 16 260 DNVFATPSPIIQQLE 16 277 DAEYTNSPLVPTFCT 16 375LSKYNSNLATPIAIK 16 394 SKRFLKHGQNIRDVS 16 118 AVNLLDKARLENQEG 15 139TKVLMEKNSMDIMKI 15 321 DLEVEDRTSLVLNSD 15 371 ILQLLSKYNSNLATP 15 1MDPIRSFCGKLRSLA 14 11 LRSLASTLDCETARL 14 22 TARLQRALDGEESDF 14 75KDDLSDPPVASSCIS 14 80 DPPVASSCISGKSPR 14 95 SPQLSDFGLERYIVS 14 100DFGLERYIVSQVLPN 14 105 RYIVSQVLPNPPQAV 14 108 VSQVLPNPPQAVNLL 14 140KVLMEKNSMDIMKIR 14 150 IMKIREYFQKYGYSP 14 163 SPRVKKNSVHEQEAI 14 168KNSVHEQEAINSDNY 14 174 QEAINSDNYKEEPVI 14 184 EEPVIVTPPTKQSLV 14 186PVIVTPPTKQSLVKV 14 197 LVKVLKTPKCALKMD 14 198 VKVLKTPKCALKMDD 14 207ALKMDDFECVTPKLE 14 217 TPKLEHFGISEYTMC 14 222 HFGISEYTMCLNEDY 14 227EYTMCLNEDYTMGLK 14 270 IQQLEKSDAEYTNSP 14 302 SIALVSTNYPLSKTN 14 303IALVSTNYPLSKTNS 14 328 TSLVLNSDTCFENLT 14 346 SPTISSYENLLRTPT 14 352YENLLRTPTPPEVTK 14 353 ENLLRTPTPPEVTKI 14 379 NSNLATPIAIKAVPP 14 385PIAIKAVPPSKRFLK 14 395 KRFLKHGQNIRDVSN 14V5-HLA-DR1-0401-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:11; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 2 DLSDPPVASSCISEK 18 3 LSDPPVASSCISEKS 18 9ASSCISEKSPRSPQL 18 5 DPPVASSCISEKSPR 14 6 PPVASSCISEKSPRS 12 11SCISEKSPRSPQLSD 12 12 CISEKSPRSPQLSDF 12 V6-HLA-DR1-0401-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:13; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 10 EEAIDAESRLNDNVF20 9 SEEAIDAESRLNDNV 18 1 LKNARNNKSEEAIDA 12 4 ARNNKSEEAIDAESR 12 6NNKSEEAIDAESRLN 12 8 KSEEAIDAESRLNDN 12 14 DAESRLNDNVFATPS 12 15AESRLNDNVFATPSP 12 V10-HLA-DR1-0401-15mers-193P1E1B Each peptide is aportion of SEQ ID NO:21; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. 13 LQKFQWIYPTQKLNK 22 15 KFQWIYPTQKLNKMR22 2 PPEVTKIPEDILQKF 20 6 TKIPEDILQKFQWIY 18 5 VTKIPEDILQKFQWI 14 10EDILQKFQWIYPTQK 14 4 EVTKIPEDILQKFQW 12 14 QKFQWIYPTQKLNKM 12V12-HLA-DR1-0401-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:25; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 6 QRALDGEESLLSKYN 26 13 ESLLSKYNSNLATPI 20 9LDGEESLLSKYNSNL 18 12 EESLLSKYNSNLATP 15 2 TARLQRALDGEESLL 14 3ARLQRALDGEESLLS 12 4 RLQRALDGEESLLSK 12 7 RALDGEESLLSKYNS 12 10DGEESLLSKYNSNLA 12 14 SLLSKYNSNLATPIA 12

TABLE XLIX Pos 123456789012345 score V1-HLA-DRB1-1101-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:3; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 159KYGYSPRVKKNSVHE 26 267 SPIIQQLEKSDAEYT 26 369 EDILQLLSKYNSNLA 26 4IRSFCGKLRSLASTL 25 349 ISSYENLLRTPTPPE 24 116 PQAVNLLDKARLENQ 22 335DTCFENLTDPSSPTI 22 194 KQSLVKVLKTPKCAL 20 233 NEDYTMGLKNARNNK 20 306VSTNYPLSKTNSSSN 20 132 GIDFIKATKVLMEKN 19 157 FQKYGYSPRVKKNSV 19 38DYPMRILYDLHSEVQ 18 105 RYIVSQVLPNPPQAV 18 300 KNSIALVSTNYPLSK 18 98LSDFGLERYIVSQVL 17 153 IREYFQKYGYSPRVK 17 191 PPTKQSLVKVLKTPK 17 210MDDFECVTPKLEHFG 17 286 VPTFCTPGLKIPSTK 17 2 DPIRSFCGKLRSLAS 16 97QLSDFGLERYIVSQV 16 144 EKNSMDIMKIREYFQ 16 186 PVIVTPPTKQSLVKV 16 287PTFCTPGLKIPSTKN 16 307 STNYPLSKTNSSSND 16 19 DCETARLQRALDGEE 15 68NFQKTDVKDDLSDPP 15 319 SNDLEVEDRTSLVLN 15 361 PPEVTKIPEDILQLL 15 381NLATPIAIKAVPPSK 15 388 IKAVPPSKRFLKHGQ 15 397 FLKHGQNIRDVSNKE 15 40PMRILYDLHSEVQTL 14 104 ERYIVSQVLPNPPQA 14 118 AVNLLDKARLENQEG 14 137KATKVLMEKNSMDIM 14 160 YGYSPRVKKNSVHEQ 14 175 EAINSDNYKEEPVIV 14 195QSLVKVLKTPKCALK 14 197 LVKVLKTPKCALKMD 14 213 FECVTPKLEHFGISE 14 214ECVTPKLEHFGISEY 14 249 EEAIDTESRLNDNVF 14 255 ESRLNDNVFATPSPI 14 358TPTPPEVTKIPEDIL 14 392 PPSKRFLKHGQNIRD 14 8 CGKLRSLASTLDCET 13 41MRILYDLHSEVQTLK 13 48 HSEVQTLKDDVNIPE 13 85 SSCISGKSPRSPQLS 13 102GLERYIVSQVLPNPP 13 127 LENQEGIDFIKATKV 13 130 QEGIDFIKATKVLME 13 136IKATKVLMEKNSMDI 13 145 KNSMDIMKIREYFQK 13 237 TMGLKNARNNKSEEA 13 293GLKIPSTKNSIALVS 13 302 SIALVSTNYPLSKTN 13 318 SSNDLEVEDRTSLVL 13 365TKIPEDILQLLSKYN 13 372 LQLLSKYNSNLATPI 13 376 SKYNSNLATPIAIKA 13 379NSNLATPIAIKAVPP 13 385 PIAIKAVPPSKRFLK 13 5 RSFCGKLRSLASTLD 12 12RSLASTLDCETARLQ 12 23 ARLQRALDGEESDFE 12 33 ESDFEDYPMRILYDL 12 57DVNIPELSNCENFQK 12 71 KTDVKDDLSDPPVAS 12 75 KDDLSDPPVASSCIS 12 82PVASSCISGKSPRSP 12 121 LLDKARLENQEGIDF 12 147 SMDIMKIREYFQKYG 12 150IMKIREYFQKYGYSP 12 165 RVKKNSVHEQEAINS 12 168 KNSVHEQEAINSDNY 12 181NYKEEPVIVTPPTKQ 12 185 EPVIVTPPTKQSLVK 12 207 ALKMDDFECVTPKLE 12 225ISEYTMCLNEDYTMG 12 232 LNEDYTMGLKNARNN 12 256 SRLNDNVFATPSPII 12 277DAEYTNSPLVPTFCT 12 282 NSPLVPTFCTPGLKI 12 291 TPGLKIPSTKNSIAL 12 350SSYENLLRTPTPPEV 12 368 PEDILQLLSKYNSNL 12 382 LATPIAIKAVPPSKR 12 383ATPIAIKAVPPSKRF 12 V5-HLA-DRB1-1101-15mers-193P1E1B Each peptide is aportion of SEQ ID NO:11; each start position is specified, the length ofpeptide is 15 amino acids, and the end position for each peptide is thestart position plus fourteen. 10 SSCISEKSPRSPQLS 13 7 PVASSCISEKSPRSP 123 LSDPPVASSCISEKS 8 8 VASSCISEKSPRSPQ 8 9 ASSCISEKSPRSPQL 8 11SCISEKSPRSPQLSD 8 6 PPVASSCISEKSPRS 7 13 ISEKSPRSPQLSDFG 7 2DLSDPPVASSCISEK 6 5 DPPVASSCISEKSPR 6 V6-HLA-DRB1-1101-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:13; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 10 EEAIDAESRLNDNVF14 13 IDAESRLNDNVFATP 7 1 LKNARNNKSEEAIDA 6 4 ARNNKSEEAIDAESR 6 6NNKSEEAIDAESRLN 6 7 NKSEEAIDAESRLND 6 14 DAESRLNDNVFATPS 6V10-HLA-DRB1-1101-15mers-193P1E1B Each peptide is a portion of SEQ IDNO:21; each start position is specified, the length of peptide is 15amino acids, and the end position for each peptide is the start positionplus fourteen. 13 LQKFQWIYPTQKLNK 16 2 PPEVTKIPEDILQKF 15 7KIPEDILQKFQWIYP 14 10 EDILQKFQWIYPTQK 12 15 KFQWIYPTQKLNKMR 11 5VTKIPEDILQKFQWI 7 9 PEDILQKFQWIYPTQ 7 V12-HLA-DRB1-1101-15mers-193P1E1BEach peptide is a portion of SEQ ID NO:25; each start position isspecified, the length of peptide is 15 amino acids, and the end positionfor each peptide is the start position plus fourteen. 10 DGEESLLSKYNSNLA20 6 QRALDGEESLLSKYN 13 13 ESLLSKYNSNLATPI 13 3 ARLQRALDGEESLLS 12

TABLE L Properties of 193P1E1B Bioinformatic Program Outcome Variants 1,5, 6 ORF ORF Finder 805-2043 Protein Length n/a 412 amino acidsTransmembrane region TM Pred No TM HMMTop No TM Sosui No TM, solubleTMHMM No TM Signal Peptide Signal P indicates no signal pI pI/MW tool pI5.03 Molecular weight pI/MW tool 46.2 kDa Localization PSORTMitochondrial 48% PSORT II Nuclear 60.9% iPSORT No signal motif MotifsPfam No motif Prints Rhodopsin Blocks No motif Prosite No motif Variant9 ORF ORF Finder 989-1981 Protein Length n/a 330 amino acidsTransmembrane region TM Pred No TM HMMTop No TM Sosui No TM, solubleTMHMM No TM Signal Peptide Signal P indicates no signal pI pI/MW tool pI5.17 Molecular weight pI/MW tool 16.5 kDa Localization PSORT Cytoplasmic45% PSORT II Nuclear 60.9% iPSORT No signal motif Motifs Pfam No motifPrints No motif Blocks No motif Prosite No motif Variant 10 ORF ORFFinder 805-1971 Protein Length n/a 388 amino acids Transmembrane regionTM Pred No TM HMMTop No TM Sosui No TM, soluble TMHMM No TM SignalPeptide Signal P indicates no signal pI pl/MW tool pl 4.8 Molecularweight pl/MW tool 34.5 kDa Localization PSORT Mitochondrial 48% PSORT IINuclear 60.9% iPSORT No signal motif Motifs Pfam No motif Prints Nomotif Blocks No motif Prosite No motif Variant 12 ORF ORF Finder805-1026 Protein Length n/a 73 amino acids Transmembrane region TM PredNo TM HMMTop No TM Sosui No TM, soluble TMHMM No TM Signal PeptideSignal P indicates no signal pI pl/MW tool pl 9.4 Molecular weight pl/MWtool 8.1 kDa Localization PSORT Mitochondrial 48% PSORT II Nuclear 60.9%iPSORT No signal motif Motifs Pfam No motif Prints No motif Blocks Nomotif Prosite No motif

TABLE LI Nucleotide sequence of transcript variant 193P1E1B v.9 (SEQ IDNO: 93) 1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaaccacaggacag 61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatatgcagactact 121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacacaagctacagtt 181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaatccaagggtga 241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctggtccgcaatga 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaaggcctccctcc 361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatgctcagccagt 421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcgcgcaacgaca 481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcggccgcgccaga 541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgtctccgcggcc 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggggcctcggcga 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggacgcgtccggcg 721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctgcgtacgcctg 781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgggaagctgcgg 841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgctggacggagag 901 gaaagcggat gatgttaata ttcttcttga taaagcaaga ttggaaaatcaagaaggcat 961 tgatttcata aaggcaacaa aagtactaat ggaaaaaaat tcaatggatattatgaaaat 1021 aagagagtat ttccagaagt atggatatag tccacgtgtc aagaaaaattcagtacacga 1081 gcaagaagcc attaactctg acccagagtt gtctaattgt gaaaattttcagaagactga 1141 tgtgaaagat gatctgtctg atcctcctgt tgcaagcagt tgtatttctgggaagtctcc 1201 acgtagtcca caactttcag attttggact tgagcggtac atcgtatcccaagttctacc 1261 aaaccctcca caggcagtga acaactataa ggaagagccc gtaattgtaaccccacctac 1321 caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaatggatgattt 1381 tgagtgtgta actcctaaat tagaacactt tggtatctct gaatatactatgtgtttaaa 1441 tgaagattac acaatgggac ttaaaaatgc gaggaataat aaaagtgaggaggccataga 1501 tacagaatcc aggctcaatg ataatgtttt tgccactccc agccccatcatccagcagtt 1561 ggaaaaaagt gatgccgaat ataccaactc tcctttggta cctacattctgtactcctgg 1621 tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaattacccattatc 1681 aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgttggttttaaa 1741 ttcagacaca tgctttgaga atttaacaga tccctcttca cctacgatttcttcttatga 1801 gaatctgctc agaacaccta cacctccaga agtaactaaa attccagaagatattctcca 1861 gcttttatca aaatacaact caaacctagc tactccaata gcaattaaagcagtgccacc 1921 cagtaaaagg ttccttaaac atggacagaa catccgagat gtcagcaacaaagaaaactg 1981 aaattccagt ggatctatcc aacacagaaa ctgaacaaaa tgagatgaaagccgagctgg 2041 accgatttta acattcacat tgccctgcct ctgtccccct ttaaacgttgacccatttta 2101 aagacaaaca tgaacattaa catcataata tgctttttat gaagtttcaataaggtttaa 2161 ccttagtctt gttgacatgt agcccagtca ttcactcttt aaggactattagtgtttcat 2221 tgatactaaa ttacccagct taatcaacag aatggtttaa gtagtaccaggaagtaggac 2281 aagtaatttc aaaaatataa aggtgtttgc tactcagatg aggccgcccctgaccttctg 2341 gccagagaga cattgctgcc agccagctct gccttcccat catctcctttcaggaccgtc 2401 ccacaccttt tacttgctca gtgctgtctg aagatgcagt tgctgtttgcaaacaacagg 2461 aacaccagtt aaactaatta ggaaacagag ggagatttcc aggcctgggtaactatatac 2521 tgtgaccatt ggcggttgag accggtcttc aaccagtgga accccgaactctgctgtcag 2581 ggtgtggact tcggtgctct tccaagtttt cacctggggg ggggagctaaccccctatgt 2641 tcacgccttc tattcccatt ggcgctgaac tcttaaggtc actctggtcgcttgtgaccc 2701 cgtaaccctg atgtacccct ctaaaaggtg aggggc

TABLE LII Nuclerotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 94)and 193P1E1B v.9 (SEQ ID NO: 95) Score = 1744 bits (907), Expect =0.0Identities = 907/907 (100%) Strand = Plus/Plus

Score = 3519 bits (1830), Expect = 0.0 Identities = 1830/1830 (100%)Strand = Plus/Plus

TABLE LIII Peptide sequences of protein coded by 193P1E1B v.9 (SEQ IDNO: 96) MEKNSMDIMK IREYFQKYGY SPRVKKNSVH EQEAINSDPE LSNCENFQKTDVKDDLSDPP  60 VASSCISGKS PRSPQLSDFG LERYIVSQVL PNPPQAVNNY KEEPVIVTPPTKQSLVKVLK 120 TPKCALKMDD FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAIDTESRLNDNV 180 FATPSPIIQQ LEKSDAEYTN SPLVPTFCTP GLKIPSTKNS IALVSTNYPLSKTNSSSNDL 240 EVEDRTSLVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDILQLLSKYNSNL 300 ATPIAIKAVP PSKRFLKHGQNIRDVSNKEN                                  330

TABLE LIV Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO: 97)and 193P1E1B v.9 (SEQ ID NO: 98) Score = 665 bits (1716), Expect= 0.0Identities = 330/330(100%), Positives = 330/330 (100%) v.1: 83MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 142MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP v.9: 1MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP  60 v.1:143 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 202VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK v.9: 61VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120 v.1:203 TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262TPKCALKMDDFECVTPKLEHEGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV v.9: 121TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 180 v.1:263 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 322FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL v.9: 181FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 240 v.1:323 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL 382EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL v.9: 241EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL 300 v.1:383 ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN                               412ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN v.9: 301ATPIAIKAVPPSKRELKHGQNIRDVSNKEN                               330

TABLE LV Nucleotide sequence of transcript variant 193P1E1B v.10 (SEQ IDNO: 99)    1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaaccacaggacag   61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatatgcagactact  121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacacaagctacagtt  181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaatccaagggtga  241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctggtccgcaatga  301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaaggcctccctcc  361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatgctcagccagt  421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcgcgcaacgaca  481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcggccgcgccaga  541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgtctccgcggcc  601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggggcctcggcga  661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggacgcgtccggcg  721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctgcgtacgcctg  781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgggaagctgcgg  841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgctggacggagag  901 gaaagcgact ttgaagatta tccaatgaga attttatatg accttcattcagaagttcag  961 actctaaagg atgatgttaa tattcttctt gataaagcaa gattggaaaatcaagaaggc 1021 attgatttca taaaggcaac aaaagtacta atggaaaaaa attcaatggatattatgaaa 1081 ataagagagt atttccagaa gtatggatat agtccacgtg tcaagaaaaattcagtacac 1141 gagcaagaag ccattaactc tgacccagag ttgtctaatt gtgaaaattttcagaagact 1201 gatgtgaaag atgatctgtc tgatcctcct gttgcaagca gttgtatttctgggaagtct 1261 ccacgtagtc cacaactttc agattttgga cttgagcggt acatcgtatcccaagttcta 1321 ccaaaccctc cacaggcagt gaacaactat aaggaagagc ccgtaattgtaaccccacct 1381 accaaacaat cactagtaaa agtactaaaa actccaaaat gtgcactaaaaatggatgat 1441 tttgagtgtg taactcctaa attagaacac tttggtatct ctgaatatactatgtgttta 1501 aatgaagatt acacaatggg acttaaaaat gcgaggaata ataaaagtgaggaggccata 1561 gatacagaat ccaggctcaa tgataatgtt tttgccactc ccagccccatcatccagcag 1621 ttggaaaaaa gtgatgccga atataccaac tctcctttgg tacctacattctgtactcct 1681 ggtttgaaaa ttccatctac aaagaacagc atagctttgg tatccacaaattacccatta 1741 tcaaaaacaa atagttcatc aaatgatttg gaagttgaag atcgtacttcgttggtttta 1801 aattcagaca catgctttga gaatttaaca gatccctctt cacctacgatttcttcttat 1861 gagaatctgc tcagaacacc tacacctcca gaagtaacta aaattccagaagatattctc 1921 cagaaattcc agtggatcta tccaacacag aaactgaaca aaatgagatgaaagccgagc 1981 tggaccgatt ttaacattca cattgccctg cctctgtccc cctttaaacgttgacccatt 2041 ttaaagacaa acatgaacat taacatcata atatgctttt tatgaagtttcaataaggtt 2101 taaccttagt cttgttgaca tgtagcccag tcattcactc tttaaggactattagtgttt 2161 cattgatact aaattaccca gcttaatcaa cagaatggtt taagtagtaccaggaagtag 2221 gacaagtaat ttcaaaaata taaaggtgtt tgctactcag atgaggccgcccctgacctt 2281 ctggccagag agacattgct gccagccagc tctgccttcc catcatctcctttcaggacc 2341 gtcccacacc ttttacttgc tcagtgctgt ctgaagatgc agttgctgtttgcaaacaac 2401 aggaacacca gttaaactaa ttaggaaaca gagggagatt tccaggcctgggtaactata 2461 tactgtgacc attggcggtt gagaccggtc ttcaaccagt ggaaccccgaactctgctgt 2521 cagggtgtgg acttcggtgc tcttccaagt tttcacctgg gggggggagctaacccccta 2581 tgttcacgcc ttctattccc attggcgctg aactcttaag gtcactctggtcgcttgtga 2641 ccccgtaacc ctgatgtacc cctctaaaag gtgaggggc

TABLE LVI Nuclerotide sequence alignment of 193P1E1B v.1 (SEQ ID NO:100) and 193P1E1B v.10 (SEQ ID NO: 101) Score = 3698 bits (1923), Expect= 0.0Identities = 1923/1923 (100%) Strand = Plus/Plus

Score = 1456 bits (757), Expect = 0.0 Identities = 757/757 (100%) Strand= Plus/Plus

TABLE LVII Peptide sequences of protein coded by 193P1E1B v.10 (SEQ IDNO: 102) MDPIRSFCGK LRSLASTLDC ETARLQRALD GEESDFEDYP MRILYDLHSEVQTLKDDVNI  60 LLDKARLENQ EGIDFIKATK VLMEKNSMDI MKIREYFQKY GYSPRVKKNSVHEQEAINSD 120 PELSNCENFQ KTDVKDDLSD PPVASSCISG KSPRSPQLSD FGLERYIVSQVLPNPPQAVN 180 NYKEEPVIVT PPTKQSLVKV LKTPKCALKM DDFECVTPKL EHFGISEYTMCLNEDYTMGL 240 KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY TNSPLVPTFCTPGLKIPSTK 300 NSIALVETNY PLSKTNSSSN DLEVEDRTSL VLNSDTCFEN LTDPSSPTISSYENLLRTPT 360 PPEVTKIPED ILQKFQWIYPTQKLNKMR                                    388

TABLE LVIII Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO:103) and 193P1E1B v.10 (SEQ ID NO: 104) Score = 749 bits (1935), Expect= 0.0Identities = 373/373 (100%), Positives = 373/373(100%) v.1:    1MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI  60           MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNIv.10:   1MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI  60v.1:   61 LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD120          LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDv.10:  61 LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD120 v.1:  121PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN 180          PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNv.10: 121 PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN180 v.1:  181NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL 240          NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLv.10: 181 NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL240 v.1:  241KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK                                               300          KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKv.10: 241KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK                                                300v.1:  301 NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT360          NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTv.10: 301 NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTTSSYENLLRTPT360 v.1:  361 PPEVTKIPEDILQ 373           PPEVTKIPEDILQ v.10: 361PPEVTKIPEDILQ 373

TABLE LIX Nucleotide sequence of transcript variant 193P1E1Bv.11 (SEQ IDNO: 105)    1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaaccacaggacag   61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatatgcagactact  121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacacaagctacagtt  181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaatccaagggtga  241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctggtccgcaatga  301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaaggcctccctcc  361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatgctcagccagt  421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcgcgcaacgaca  481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcggccgcgccaga  541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgtctccgcggcc  601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggggcctcggcga  661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggacgcgtccggcg  721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctgcgtacgcctg  781 gagtccttcc ccgctgtgct cagcatggac catatccgga gcttctgcgggaagctgcgg  841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgctggacggagag  901 gaaagcggat gatgttaata ttcttcttga taaagcaaga ttggaaaatcaagaaggcat  961 tgatttcata aaggcaacaa aagtactaat ggaaaaaaat tcaatggatattatgaaaat 1021 aagagagtat ttccagaagt atggatatag tccacgtgtc aagaaaaattcagtacacga 1081 gcaagaagcc attaactctg acccagagtt gtctaattgt gaaaattttcagaagactga 1141 tgtgaaagat gatctgtctg atcctcctgt tgcaagcagt tgtatttctgggaagtctcc 1201 acgtagtcca caactttcag attttggact tgagcggtac atcgtatcccaagttctacc 1261 aaaccctcca caggcagtga acaactataa ggaagagccc gtaattgtaaccccacctac 1321 caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaatggatgattt 1381 tgagtgtgta actcctaaat tagaacactt tggtatctct gaatatactatgtgtttaaa 1441 tgaagattac acaatgggac ttaaaaatgc gaggaataat aaaagtgaggaggccataga 1501 tacagaatcc aggctcaatg ataatgtttt tgccactccc agccccatcatccagcagtt 1561 ggaaaaaagt gatgccgaat ataccaactc tcctttggta cctacattctgtactcctgg 1621 tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaattacccattatc 1681 aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgttggttttaaa 1741 ttcagacaca tgctttgaga atttaacaga tccctcttca cctacgatttcttcttatga 1801 gaatctgctc agaacaccta cacctccaga agtaactaaa attccagaagatattctcca 1861 gaaattccag tggatctatc caacacagaa actgaacaaa atgagatgaaagccgagctg 1921 gaccgatttt aacattcaca ttgccctgcc tctgtccccc tttaaacgttgacccatttt 1981 aaagacaaac atgaacatta acatcataat atgcttttta tgaagtttcaataaggttta 2041 accttagtct tgttgacatg tagcccagtc attcactctt taaggactattagtgtttca 2101 ttgatactaa attacccagc ttaatcaaca gaatggttta agtagtaccaggaagtagga 2161 caagtaattt caaaaatata aaggtgtttg ctactcagat gaggccgcccctgaccttct 2221 ggccagagag acattgctgc cagccagctc tgccttccca tcatctcctttcaggaccgt 2281 cccacacctt ttacttgctc agtgctgtct gaagatgcag ttgctgtttgcaaacaacag 2341 gaacaccagt taaactaatt aggaaacaga gggagatttc caggcctgggtaactatata 2401 ctgtgaccat tggcggttga gaccggtctt caaccagtgg aaccccgaactctgctgtca 2461 gggtgtggac ttcggtgctc ttccaagttt tcacctgggg gggggagctaaccccctatg 2521 ttcacgcctt ctattcccat tggcgctgaa ctcttaaggt cactctggtcgcttgtgacc 2581 ccgtaaccct gatgtacccc tctaaaaggt gaggggc

TABLE LX Nucteotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 106)and 193P1E1B v.11 (SEQ ID NO: 107) Score = 1744 bits (907), Expect =0.0Identities = 907/907 (100%) Strand = Plus/Plus

Score = 1836 bits (955), Expect = 0.0Identities = 955/955 (100%) Strand= Plus/Plus

Score = 1456 bits (757), Expect = 0.0Identities = 757/757 (100%) Strand= Plus/Plus

TABLE LXI Peptide sequences of protein coded by 193P1E1B v.11 (SEQ IDNO: 108) MEKNSMDIMK IREYFQKYGY SPRVKKNSVH EQEATNSDPE LSNCENFQKTDVKDDLSDPP  60 VASSCISGKS PRSPQLSDFG LERYIVSQVL PNPPQAVNNY KEEPVIVTPPTKQSLVKVLK 120 TPKCALKMDD FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAIDTESRLNDNV 180 FATPSPIIQQ LEKSDAEYTN SPLVPTFCTP GLKIPSTKNS IALVSTNYPLSKTNSSSNDL 240 EVEDRTSLVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDILQKFQWIYPTQ 300KLNKMR                                                            306

TABLE LXII Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO:109) and 193P1E1B v.11 (SEQ ID NO: 110) Score = 589 bits (1518), Expect= e-167Identities = 291/291 (100%), Positives = 291/291 (100%) v.1:   83MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 142          MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPv.11:   1MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP  60v.1:  143 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK202          VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKv.11:  61 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK120 v.1:  203TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262          TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVv.11: 121 TPKCALKMDDFECVTPKLEHFGTSEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV180 v.1:  263FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 322          FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKTPSTKNSIALVSTNYPLSKTNSSSNDLv.11: 181 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL240 v.1:  323EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ          373          EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ v.11: 241EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLITPTPPEVTKIPEDILQ          291

TABLE LXIII Nucleotide sequence of transcript variant 193P1E1B v.12 (SEQID NO: 111)    1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaaccacaggacag   61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatatgcagactact  121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacacaagctacagtt  181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaatccaagggtga  241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctggtccgcaatga  301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaaggcctccctcc  361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatgctcagccagt  421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcgcgcaacgaca  481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcggccgcgccaga  541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgtctccgcggcc  601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggggcctcggcga  661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggacgcgtccggcg  721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctgcgtacgcctg  781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgggaagctgcgg  841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgctggacggagag  901 gaaagccttt tatcaaaata caactcaaac ctagctactc caatagcaattaaagcagtg  961 ccacccagta aaaggttcct taaacatgga cagaacatcc gagatgtcagcaacaaagaa 1021 aactgaaatt ccagtggatc tatccaacac agaaactgaa caaaatgagatgaaagccga 1081 gctggaccga ttttaacatt cacattgccc tgcctctgtc cccctttaaacgttgaccca 1141 ttttaaagac aaacatgaac attaacatca taatatgctt tttatgaagtttcaataagg 1201 tttaacctta gtcttgttga catgtagccc agtcattcac tctttaaggactattagtgt 1261 ttcattgata ctaaattacc cagcttaatc aacagaatgg tttaagtagtaccaggaagt 1321 aggacaagta atttcaaaaa tataaaggtg tttgctactc agatgaggccgcccctgacc 1381 ttctggccag agagacattg ctgccagcca gctctgcctt cccatcatctcctttcagga 1441 ccgtcccaca ccttttactt gctcagtgct gtctgaagat gcagttgctgtttgcaaaca 1501 acaggaacac cagttaaact aattaggaaa cagagggaga tttccaggcctgggtaacta 1561 tatactgtga ccattggcgg ttgagaccgg tcttcaacca gtggaaccccgaactctgct 1621 gtcagggtgt ggacttcggt gctcttccaa gttttcacct gggggggggagctaaccccc 1681 tatgttcacg ccttctattc ccattggcgc tgaactctta aggtcactctggtcgcttgt 1741 gaccccgtaa ccctgatgta cccctctaaa aggtgagggg c

TABLE LXIV Nucleotide sequence alignment of 193P1E1B v.1 (SEQ ID NO:112) and 193P1E1B v.12 (SEQ ID NO: 113) Score = 1742 bits (906), Expect= 0.0Identities = 906/906 (100%) Strand = Plus/Plus

Score = 1683 bits (875), Expect = 0.0Identities = 875/875 (100%) Strand= Plus/Plus

TABLE LXV Peptide sequences of protein coded by 193P1E1B v.12 (SEQ IDNO: 114) MDPIRSFCGK LRSLASTLDC ETARLQRALD GEESLLSKYN SNLATPIAIKAVPPSKRFLK 60 HGQNIRDVSNKEN                                                          73

TABLE LXVI Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO:115) and 193P1E1B v.12 (SEQ ID NO: 116) Score = 72.0 bits (175), Expect= 2e-12Identities = 35/39 (89%), Positives = 35/39 (89%)v.1:        1      MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDY 39                   MDPIRSECGKLRSLASTLDCETARLQRALDGEES    Yv.12:       1      MDPIRSFCGKLRSLASTLDCETARLQRALDGEESLLSKY 39 Score= 80.9 bits (198), Expect = 4e-15Identities = 39/39 (100%), Positives= 39/39 (100%)v.1:      374      LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 412                   LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKENv.12:      35      LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN  73

TABLE LXVII Nucleotide sequence of transcript variant 193P1E1B v.13 (SEQID NO: 117)    1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaaccacaggacag   61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatatgcagactact  121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacacaagctacagtt  181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaatccaagggtga  241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctggtccgcaatga  301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaaggcctccctcc  361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatgctcagccagt  421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcgcgcaacgaca  481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcggccgcgccaga  541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgtctccgcggcc  601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggggcctcggcga  661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggacgcgtccggcg  721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctgcgtacgcctg  781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgggaagctgcgg  841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgctggacggagag  901 gaaagcggtg cgtgaggcgg gcggccaggg cacgactttg aagattatccaatgagaatt  961 ttatatgacc ttcattcaga agttcagact ctaaaggatg atgttaatattcttcttgat 1021 aaagcaagat tggaaaatca agaaggcatt gatttcataa aggcaacaaaagtactaatg 1081 gaaaaaaatt caatggatat tatgaaaata agagagtatt tccagaagtatggatatagt 1141 ccacgtgtca agaaaaattc agtacacgag caagaagcca ttaactctgacccagagttg 1201 tctaattgtg aaaattttca gaagactgat gtgaaagatg atctgtctgatcctcctgtt 1261 gcaagcagtt gtatttctgg gaagtctcca cgtagtccac aactttcagattttggactt 1321 gagcggtaca tcgtatccca agttctacca aaccctccac aggcagtgaacaactataag 1381 gaagagcccg taattgtaac cccacctacc aaacaatcac tagtaaaagtactaaaaact 1441 ccaaaatgtg cactaaaaat ggatgatttt gagtgtgtaa ctcctaaattagaacacttt 1501 ggtatctctg aatatactat gtgtttaaat gaagattaca caatgggacttaaaaatgcg 1561 aggaataata aaagtgagga ggccatagat acagaatcca ggctcaatgataatgttttt 1621 gccactccca gccccatcat ccagcagttg gaaaaaagtg atgccgaatataccaactct 1681 cctttggtac ctacattctg tactcctggt ttgaaaattc catctacaaagaacagcata 1741 gctttggtat ccacaaatta cccattatca aaaacaaata gttcatcaaatgatttggaa 1801 gttgaagatc gtacttcgtt ggttttaaat tcagacacat gctttgagaatttaacagat 1861 ccctcttcac ctacgatttc ttcttatgag aatctgctca gaacacctacacctccagaa 1921 gtaactaaaa ttccagaaga tattctccag cttttatcaa aatacaactcaaacctagct 1981 actccaatag caattaaagc agtgccaccc agtaaaaggt tccttaaacatggacagaac 2041 atccgagatg tcagcaacaa agaaaactga aattccagtg gatctatccaacacagaaac 2101 tgaacaaaat gagatgaaag ccgagctgga ccgattttaa cattcacattgccctgcctc 2161 tgtccccctt taaacgttga cccattttaa agacaaacat gaacattaacatcataatat 2221 gctttttatg aagtttcaat aaggtttaac cttagtcttg ttgacatgtagcccagtcat 2281 tcactcttta aggactatta gtgtttcatt gatactaaat tacccagcttaatcaacaga 2341 atggtttaag tagtaccagg aagtaggaca agtaatttca aaaatataaaggtgtttgct 2401 actcagatga ggccgcccct gaccttctgg ccagagagac attgctgccagccagctctg 2461 ccttcccatc atctcctttc aggaccgtcc cacacctttt acttgctcagtgctgtctga 2521 agatgcagtt gctgtttgca aacaacagga acaccagtta aactaattaggaaacagagg 2581 gagatttcca ggcctgggta actatatact gtgaccattg gcggttgagaccggtcttca 2641 accagtggaa ccccgaactc tgctgtcagg gtgtggactt cggtgctcttccaagttttc 2701 acctgggggg gggagctaac cccctatgtt cacgccttct attcccattggcgctgaact 2761 cttaaggtca ctctggtcgc ttgtgacccc gtaaccctga tgtacccctctaaaaggtga 2821 ggggc

TABLE LXVIII Nucleotide sequence alignment of 193P1E1B v.1 (SEQ ID NO:118) and 193P1E1B v.13 (SEQ ID NO: 119) Score 1744 bits (907), Expect =0.0Identities = 907/907 (100%) Strand = Plus/Plus

Score = 3640 bits (1893), Expect = 0.0Identities = 1893/1893 (100%)Strand = Plus/Plus

Score = 1744 bits (907), Expect = 0.0Identities = 907/907 (100%) Strand= Plus/Plus

TABLE LXIX Peptide sequences of protein coded by 193P1E1B v.13 (SEQ IDNO:120) MRILYDLHSE VQTLKDDVNI LLDKARLENQ EGIDFIKATK VLMEKNSMDIMKIREYFQKY  60 GYSPRVKKNS VHEQEAINSD PELSNCENFQ KTDVKDDLSD PPVASSCISGKSPRSPQLSD 120 FGLERYIVSQ VLPNPPQAVN NYKEEPVIVT PPTKQSLVKV LKTPKCALKMDDFECVTPKL 180 EHFGISEYTM CLNEDYTMGL KNARNNKSEE AIDTESRLND NVFATPSPIIQQLEKSDAEY 240 TNSPLVPTFC TPGLKIPSTK NSIALVSTNY PLSKTNSSSN DLEVEDRTSLVLNSDTCFEN 300 LTDPSSPTIS SYENLLRTPT PPEVTKIPED ILQLLSKYNS NLATPIAIKAVPPSKRFLKH 360 GQNIRDVSNK EN 372

TABLE LXX Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO:121)and 193P1E1B v.13 (SEQ ID NO:122) Score = 745 bits (1923), Expect= 0.0Identities = 372/372 (100%), Positives = 372/372 (100%) V.1: 41MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY 100MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY V.13: 1MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY  60 V.1:101 GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD 160GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD V.13: 61GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD 120 V.1:161 FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL 220FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL V.13: 121FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL 180 V.1:221 EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY 280EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY V.13: 181EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY 240 V.1:281 TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN 340TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN V.13: 241TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN 300 V.1:341 LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH 400LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH V.13: 301LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH 360 V.1:401 GQNIRDVSNKEN 412 GQNIRDVSNKEN V.13: 361 GQNIRDVSNKEN 372

1. A method for detecting, in a sample, the presence of a protein,comprising: contacting the sample with an antibody or fragment thereofthat specifically binds to a protein comprising the amino acid sequenceof SEQ ID NO:3, SEQ ID NO: 11 or SEQ ID NO: 13; and, detecting theformation and/or presence of a complex comprising the antibody and theprotein, wherein the protein comprises an amino acid sequence, andwherein the amino acid sequence is selected from group consisting of SEQID NO:3, SEQ ID NO: 11, and SEQ ID NO:
 13. 2. The method of claim 1,wherein the protein comprises the amino acid sequence of SEQ ID NO: 11or SEQ ID NO: 13.